O estudo fitoquímico de Peperomia oreophila revelou a presençca de duas lignanas furofurânicas (7R, 8R, 7R, 8R)-3,4,5-trimetóxi-3,4-metilenodioxi-5-metóxi- 8.8,7.O.9,7.O.9-lignana (1), (7R, 8R, 7R, 8R)-3,4,5-trimetóxi-3,4,5-trimetóxi- 8.8,7.O.9,7.O.9-lignana (2); as duas amidas (2E)-N-isobutil-3-(5-metóxi-7,8- benzodioxol-1-il)acrilamida (3), (2E)-N-isobutil-3-(3,4,5-trimetóxifenil)acrilamida (4), três derivados de acido cinâmico (2E)-3-(3,4,5-trimetóxifenil)acrilato de metila (5), (2Z)-3- (3,4,5-trimetóxifenil)acrilato de metila (6), (2E)-3-(5-metóxi-7,8-benzodioxol-1-il)acrilato de metila (7); os dois policetídeos fenólicos [(2E)-3,7-dimetilocta-2,6-dien-1-il]-5- metil-2-(3-metilbut-2-en-1-il)benzeno-1,3-diol (8) (inédita) e [(2E)-3,7-dimetilocta- 2,6-dien-1-il]-2,2,7-trimetil-2H-cromen-5-ol (9); de P. arifolia: o policetídeo fenólico [(2E)-3,7-dimetilocta-2,6-dien-1-il]-5-metil-2-(3-metilbut-2-en-1-il) benzeno-1,3-diol, isolada também de P. oreophila (10) (inédita); de P. urocarpa: o policetídeo fenólico 5- metil-2-[(2E,6E)-3,7,11-trimetildodeca-2,6,10-trien-1-il] benzeno-1,3-diol (11) e o ácido 2,4-dihidróxi-6-metil-3-[(2E,6E)-3,7,11-trimetildodeca-2,6,10-trien-1-il] benzóico, (12); de P. nitida: o fenilpropanoide apiol (1-alil-3,6-dimetóxi-10,11- benzodioxol) (13), os cromenos 7-hidróxi-2,2,5-trimetil-2H-cromeno-carboxilato de metila (14) e o 7-metóxi-2,2,5-dimetil-2H-cromeno-6-carboxilato de metila (15). O policetídeo 2-hidróxi-4,6-dimetóxiacetofenona, principal metabólito das folhas de P. glabella, teve sua biossíntese investigada utilizando-se como precursores o acetil-CoA e o malonil-CoA. Foram realizados estudos de otimização da atividade de policetídeo sintase (PKS) em função do pH, tempo de reação, temperatura e saturação de substratos, além de estudos da variação circadiana. Estudos de genes de PKS resultaram em amplificações cujo seqüenciamento poderá determinar a identidade dessas regiões e homologia entre as seqüências dessas Peperomias e a região KS do gene AviM de Streptomyces viridochromogenes que expressa o ácido orselínico / The phytochemical investigation carried out on Peperomia oreophila revealed the accumulation of two furofuran lignans (7R,8R,7R,8R)-3,4,5-trimethoxy-3,4- methylenedioxy-8.8, 7.O.9, 9.O.7-lignan (1), (7, 8R, 7R, 8)-3,4,5-trimethoxy-3,4,5- trimethoxy-8.8-7.O.9, 9.O.7-lignan (2); two amides (2´E)-N-isobutyl-3´-(5-methoxy-7,8- benzodioxol-1-yl) acrylamide (3), (2E)-N-isobutyl-3-(3,4,5-trimethoxyphenyl)acrylamide (4), three derivate cinâmic acid methyl (2E)-3-(3,4,5-trimethoxyphenyl)acrylate (5), methyl (2Z)-3-(3,4,5-trimethoxyphenyl)acrylate (6), methyl (2E)-3-(5-methoxy-7,8- benzodioxol-1-yl)acrylate (7); two phenolic polyketides [(2E)-3,7-dimethylocta-2,6- dien-1-yl]-5-methyl-2-(3-methylbut-2-en-1-yl)benzene-1,3-diol (8) (novel), [(2´E)-3´,7´- dimethylocta-2´,6´-dien-1-yl]-2,2,7-trimethyl-2H-chromen-5-ol (9); P. arifolia, two phenolic polyketide [(2E)-3,7-dimethylocta-2,6-dien-1-yl]-5-methyl-2-(3-methylbut-2- en-1-yl)benzene-1,3-diol, also isolated from P. oreophila (10) (novel); P. urocarpa, the two phenolic polyketides 5-methyl-2-[(2E,6E)-3,7,11-trimethyldodeca-2,6,10-trien-1- yl]benzene-1,3-diol (11) and 2,4-dihydroxy-6-methyl-3-[(2E,6E)-3,7,11-trimethyldodeca- 2,6,10-trien-1-yl]benzoic acid (12); P. nitida, the phenylpropanoid apiole 1-allyl-3,6- dimethoxy-10,11-benzodioxole (13); the chromenes methyl 7-hydroxy-2,2,5-trimethyl- 2H-chromene-6-carboxylate (14) and methyl 7-methoxy-2,2,5-trimethyl-2H-chromene-6- carboxylate (15). The polyketide 2-hydroxy-4,6-dimethoxyacetophenone, the major compound in P. glabella leaves, had its biosynthetic origin investigated using acetyl-CoA and malonyl-CoA as precursors. The enzymatic activity of polyketide synthase was optimized to pH, incubation time, temperatures and substrate saturation, in addition to the analysis of circadian variation activity. The amplifications of putative PKS genes was based on primers from AviM gene of Streptomyces viridochromogenes that express for orsellinic acid. The sequencing will enable the identification of such regions and also to study the homology to fungi PKS

The soil bacterium Rhodococcus is a member of the phylum Actinobacteria and is related to Streptomyces, which is known for its production of many secondary metabolites. Recent genomic investigation of Rhodococcus has uncovered many silent gene clusters that appear to code for nonribosomal peptide synthetases (NRPSs) and polyketide synthases (PKS) of unknown function. Previous work, showed that Rhodococcus species strain KCHXC3 produces an inhibitory compound in agar culture extracts that displays prominent activity against several Gram positive and Gram negative species including the pathogens Rhodococcus equi, Shigella dysenteriae and Pseudomonas aeruginosa. Using the engineered Rhodococcus transposon vector, pTNR, the goal of this investigation is to screen random mutants of KCHXC3 for strains that no longer produce the inhibitory molecule. A library of 1825 random insertion mutants was produced via electroporation then screened for production of the inhibitory molecule by a disk diffusion assay against Shigella dysenteriae. From this screening, 7 mutants which no longer produce the compound of interest were identified.

Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

In this study, novel polyketide biosynthesis gene cluster of Aspergillus fumigatus was characterized and functionally analyzed. Analysis of the newly sequenced A. fumigatus genome for laccases, which are involved in melanin biosynthesis and detoxification in fungi, resulted in several putative laccase and multicopper oxidase gene sequences, one of which, Afu4g14490 (tpnJ), was selected for further characterization. The predicted amino acid sequence TpnJp showed 63% identity with the dihydrogeodin oxidase of Aspergillus terreus, which is involved in the biosynthesis of the antifungal geodin. When the genome region of tpnJ was investigated, the presence of a polyketide biosynthesis gene cluster containing 13 genes, hypothesized to be responsible for the production of trypacidin and monomethylsulochrin, was realized. By a comparative genomics approach, a putative geodin biosynthesis gene cluster containing 13 genes, including dihydrogeodin oxidase, in A. terreus and a putative trypacidin biosynthesis gene cluster containing 13 genes in N. fischeri were established. Targeted deletions of the polyketide synthase (tpnC) and multicopper oxidase (tpnJ) genes confirmed the hypothesis that TpnCp, a three-domain minimal polyketide synthase, is involved in trypacidin and monomethylsulochrin biosynthesis in A. fumigatus. TpnCp is the first fungal minimal polyketide synthase whose functional role was experimentally identified. Moreover, the fact that LC-MS analysis of DtpnJ strain showed the absence of trypacidin and the presence of a higher amount of monomethylsulochrin in DtpnJ strain, confirmed the hypothesis that TpnJp is involved in the oxidation of monomethylsulochrin into trypacidin. This novel multicopper oxidase having high substrate specificity is given the name monomethylsulochrin oxidase.

Cryptosporidium parvum is widely known for outbreaks within the immunocompetent population, as well its sometimes excruciating effects as an opportunistic agent in AIDS patients. Our understanding of the biology and host-parasite interactions of this parasitic protist is increasing at a rapid rate due to recent molecular and genetic advances. The topic of our research is in the area of C. parvum fatty acid metabolism, which is highly streamlined in this parasite. In addition to a type I fatty acid synthase (CpFAS1), C. parvum also possesses an enormous type I polyketide synthase (CpPKS1). Because of the size of this megasynthase, functional characterization of the complete enzyme is not possible. We have isolated and characterized the loading unit of CpPKS1 which contains an acyl-[acyl carrier protein (ACP)] ligase (AL) and an ACP. This unit is responsible for the overall substrate selection and initiation of polyketide production. Our data show that CpPKS1 prefers long-chain fatty acids with the highest specificity for arachidic acid (C20). Thus, the final polyketide product could contain as many as 34 carbons. Additionally, C. parvum possesses only a single fatty acid elongase. This family of enzymes serves a mechanism similar to FAS, and many have been found to be involved in de novo fatty acid synthesis in other organisms. After expressing this membrane protein in human cells, we have determined that it too prefers long-chain fatty acyl-CoAs which undergo only one round of elongation. This is in contrast to members of this enzyme family in other organisms that can initiate de novo synthesis from two- or four-carbon fatty acids via several rounds of elongation. Our lab has previously characterized the unique acyl-CoA binding protein (CpACBP1) from C. parvum. Molecular and biochemical data suggested that this enzyme may serve as a viable drug target. We have screened a library of known (and somewhat common) compounds against CpACBP1, and have isolated several potential compounds to be further examined for their ability to inhibit the growth of C. parvum.

Under the conditions of transfer hydrogenation utilizing chromatographically purified ortho-cyclometallated iridium C,O-benzoate precatalysts, enantioselective carbonyl crotylation and methallylation can be performed in the absence of stoichiometric metallic reagents and stoichiometric chiral modifiers. In the case of carbonyl crotylation, use of a preformed precatalyst rather than an in situ generated catalyst results in lower reaction temperatures, providing generally higher diastereoselectivity and yields. By utilizing a more reactive leaving group in chloride over acetate on our methallyl donor, the inherently shorter lifetime of the olefin π-complex is compensated for, giving our group’s first report of reactivity utilizing 1,1-disubstituted allyl donors. / text

Modular polyketide synthases (PKSs) are the largest enzymes known to man and are responsible for synthesizing some of the most important human medicines. Their ability to construct stereochemically-rich carbon chains containing diverse substituents has inspired the biosynthetic community to engineer these factories for the in vitro synthesis of a small library of polyketide compounds. New complex polyketides are discovered every year, yet the lack of compound prohibits characterization and testing of these new compounds for medicinal properties. Smaller polyketide compounds generated in vitro could be organically manipulated to generate larger, more complex polyketide natural products and natural product analogs. Chemoenzymatic approaches like this would be extremely beneficial to the scientific community; however, there are still obstacles that must be overcome before the use of PKS for the preparative synthesis of an in vitro generated polyketide library would prove fruitful: purchasing substrates such as methylmalonyl-CoA is cost-prohibitive, PKSs are often difficult to express and purify, and the products generated are typically nonchromophoric. The use of a malonyl-CoA ligase from Streptomyces coelicolor (MatB) was investigated for the enzymatic synthesis of polyketide extender units such as methylmalonyl-CoA (Chapter 2). MatB synthesized a total of 5 CoA-linked extender units in vitro: malonyl-, methylmalonyl-, ethylmalonyl-, hydroxymalonyl- and methoxymalonyl-CoA. Two ternary complex structures of MatB with bound product and leaving group were also solved to sub-2Å resolution. MatB generated extender units were employed in the module-catalyzed synthesis of a triketide pyrone. The selectivity of a PKS module to incorporate a variety of side chains into triketide pyrones was also investigated (Chapter 3). A total of 10 triketide pyrone compounds were synthesized, 5 produced via modular "stuttering" and one possessing a terminal alkyne chemical handle. Lastly, nonchromphoric polyketide products were made visible upon copper(I)-catalyzed azide alkyne cycloaddition (CuAAC) with fluorescent sulforhodamine B azide revealing insights into in vitro reactivites of a PKS module (Chapter 4). The work described in this dissertation has helped advance the scientific community towards procuring an in vitro synthesized polyketide library for future synthetic applications. / text

This dissertation includes molecular and phytochemical investigations of the harmful, bloom-forming alga, Prymnesium parvum, including analysis of known polyketide metabolites as a function of salinity and growth. Initially, the development of molecular and phytochemical tools was necessary for the detection and quantification of P. parvum and its associated toxins. Suites of oligonucleotides and molecular beacons were designed for conventional and quantitative multiplex PCR to amplify four species- and gene-specific products simultaneously that were used for the detection and quantitation of P. parvum. This built-in redundancy provided increased confidence in reactions with the positive confirmation of four discrete products. Techniques were also developed for the chemical enrichment of toxins produced by P. parvum. Until now, isolation of “prymnesins” has never been reproduced. Polyketide prymnesins possess unique spectral properties that were used to generate an LC-MS fingerprint that comprised 13 ion species. Preliminary investigations using chemifluorimetric methods were also capable of detecting prymnesins in the pico- and nano-molar range. Environmental samples were tested as an independent assessment of these methods. Lastly, the roles of polyketide prymnesins were analyzed with respect to total hemolytic activity (HA) as a function of culture age and salinity. Variation in HA of supernatants was statistically significant relative to both variables (p << 0.05). Salinity was inversely related to HA wherein cultures growing in 5-25 psu were 150-200% more hemolytic. Total HA was inversely related to culture age during the first three weeks, but positively related to it during the next three weeks. Interestingly, no hemolysis was detected in fractions containing prymnesins from culture supernatants and the majority of hemolysins remained in the aqueous phase. Prymnesins extracted from cells varied significantly over the 6-week observation period (p << 0.05); HA was positively correlated during the first half and inversely related during the last half of the study. Salinity was directly related to HA from cell extracts, but these effects were not significantly different until the last three weeks. These investigations suggest that polyketide prymnesins are present at much lower quantities than previously believed, and they may not be the key compounds associated with hemolysis due to P. parvum. / text

Type II polyketide synthases (PKSs) are responsible for the generation of structurally diverse and clinically important aromatic polyketides. The bacteriostatic agent enterocin (enc) isolated from the marine microbe "Streptomyces maritimus" is derived from a rare benzoate primer unit and contains a unique nonaromatic caged core structure resulting from a Favorskii-like carbon skeleton rearrangement. The apparent diversion between enterocin biosynthesis and all other type II PKS pathways offered the opportunity to discover novel enzymatic strategies that may be exploited to diversify the chemical structures of polyketides. A comprehensive biochemical analysis was performed in order to characterize the key steps in enterocin biosynthesis and finally to reconstitute the whole pathway in vitro using purified recombinant enzymes.A nonribosomal peptide synthetase (NRPS)-like priming mechanism was discovered for the selective activation of a benzoic acid starter unit and its subsequent attachment to the enc PKS to initiate polyketide biosynthesis. This is the first example of a type II PKS that employs an NPRS-like priming mechanism to utilize alternative non- acetate starter units. Secondly, the minimal enc PKS was reconstituted in vitro to give three novel acetate-primed metabolites that had never been identified by heterologous in vivo expression of recombinant enc PKS gene sets. The minimal enc PKS was then merged with the NRPS-like chain initiation module and the resulting multienzyme complex catalyzed the formation of benzoate-primed natural products wailupemycin F and wailupemycin G. Favorskii-like rearrangement of the nascent polyketide chain was replicated in vitro and the flavin-dependent oxygenase EncM was confirmed to be solely responsible for catalyzing this unprecedented rearrangement. Other biosynthetic steps in the late stage of the enc pathway were also replicated in vitro, including the methylation of desmethyl-5-deoxyenterocin to 5-deoxyenterocin and the hydroxylation of 5-deoxyenterocin to enterocin.Finally, the entire enc type II PKS pathway was successfully assembled in vitro using ten recombinant proteins and three commercial enzymes. Five enc-based natural products were generated from benzoic acid and malonyl-coenzyme A. This biochemical investigation on enterocin biosynthesis represents the first complete in vitro reconstitution of a type II PKS system and also provides an alternative strategy to create complex natural products by multienzyme synthesis.

Fungal polyketides are synthesized by polyketide synthases (PKS) encoded by PKS genes. The function of many PKS genes is unknown and the number of PKS genes exceeds the number of polyketides in many genomes. The lichen-forming fungi, Cladonia and Ramalina have chemical variants separated by habitat suggesting that environmental conditions may influence polyketide production. The goal of this thesis was to examine evolutionary relationships as a framework to investigate PKS gene function in the lichen-forming fungal families Cladoniaceae and Ramalinaceae. A phylogenetic analysis of the genus Ramalina (Chapter 2) using nuclear and mitochondrial ribosomal DNA sequences showed monophyly for seven species and included three species, which were not examined in phylogenies prior to this study. One monophyletic species, R. dilacerata was chosen for further tests of the effect of growing conditions on PKS gene expression (Chapter 3). Growth media containing yeast extracts produced the largest colony diameters and the fewest number of polyketides. A significant negative relationship occurred between colony diameter and number of secondary metabolites. Expression of two types of PKS genes was correlated with pH-level and media conditions that produced larger numbers of secondary products in R. dilacerata. A PKS gene phylogeny was constructed for 12 paralogs detected in members of the C. chlorophaea complex (Chapter 4) and gene selection was inferred using dN/dS estimations. The gene phylogeny provided evidence for independent origins and purifying and positive selection of PKS paralogs. This research provided insight into the evolution of PKS genes in the C. chlorophaea complex and identified potential genes that produce non-reduced polyketides present in C. chlorophaea. This thesis provided evidence for diversification of both morphological and chemical species and monophyly of previously unstudied Ramalina species. This research also supported theories of secondary metabolite synthesis based on growing conditions of R. dilacerata, and it revealed that PKS genes under selection in the Cladonia chlorophaea group provide the lichen with the adaptive capacity to survive under variable conditions. Knowledge of the ecological function of fungal polyketides can be valuable for conservation management and policy makers; and for understanding the potential pharmaceutical roles of these natural products.