Functional analysis of Hypoxylon pulicicidum genes required for heterologous biosynthesis of nodulisporic acids

<p>Nature holds some of the greatest secrets in drug design and development and the ability to access these trade secrets has been revolutionised by modern bioengineering technologies. In order to exploit these technologies it is essential to understand what genes are involved in compound production and the enzymatic steps that limit flux to the desired product. This thesis describes the discovery of four secondary-metabolic enzymatic steps involved in the biosynthesis of a group of valuable natural products known as nodulisporic acids. Nodulisporic acids are known for their potent insecticidal activities; however, biosynthesis of these compounds by the natural fungal producer, Hypoxylon pulicicidum (Nodulisporium sp.), is exceptionally difficult and has prevented the commercial development of novel nodulisporic acid-containing veterinary medicines and crop protects. To discover how nodulisporic acids are biosynthesized: 1. the H. pulicicidum genome was sequenced 2. a gene cluster responsible for nodulisporic acid production was predicted 3. genes in the cluster were functionally characterised by pathway reconstitution in a common, fast growing mould, Penicillium paxilli In turn, four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F, have been functionally characterised. The four genes encode a geranylgeranyl transferase (NodC), a flavin adenine dinucleotide-dependent oxygenase (NodM), an indole diterpene cyclase (NodB) and a cytochrome P450 oxygenase (NodW). Two of the gene products (NodM and NodW) catalyse two previously unreported reactions that provide the enzymatic basis of the biosynthetic branch point unique to nodulisporic acid biosynthesis. From here, future efforts will explore how these genes can be engineered to overcome flux bottlenecks and enable production of significantly increased, and even industrially relevant, product titres.</p>

Functional analysis of Hypoxylon pulicicidum genes required for heterologous biosynthesis of nodulisporic acids

<p>Nature holds some of the greatest secrets in drug design and development and the ability to access these trade secrets has been revolutionised by modern bioengineering technologies. In order to exploit these technologies it is essential to understand what genes are involved in compound production and the enzymatic steps that limit flux to the desired product. This thesis describes the discovery of four secondary-metabolic enzymatic steps involved in the biosynthesis of a group of valuable natural products known as nodulisporic acids. Nodulisporic acids are known for their potent insecticidal activities; however, biosynthesis of these compounds by the natural fungal producer, Hypoxylon pulicicidum (Nodulisporium sp.), is exceptionally difficult and has prevented the commercial development of novel nodulisporic acid-containing veterinary medicines and crop protects. To discover how nodulisporic acids are biosynthesized: 1. the H. pulicicidum genome was sequenced 2. a gene cluster responsible for nodulisporic acid production was predicted 3. genes in the cluster were functionally characterised by pathway reconstitution in a common, fast growing mould, Penicillium paxilli In turn, four genes involved in the biosynthesis of the nodulisporic acid core compound, nodulisporic acid F, have been functionally characterised. The four genes encode a geranylgeranyl transferase (NodC), a flavin adenine dinucleotide-dependent oxygenase (NodM), an indole diterpene cyclase (NodB) and a cytochrome P450 oxygenase (NodW). Two of the gene products (NodM and NodW) catalyse two previously unreported reactions that provide the enzymatic basis of the biosynthetic branch point unique to nodulisporic acid biosynthesis. From here, future efforts will explore how these genes can be engineered to overcome flux bottlenecks and enable production of significantly increased, and even industrially relevant, product titres.</p>

Avaliação microbiológica de ovos revestidos com nanobiopolímero a base de isolado proteico de soro

O ovo é um alimento proteico barato e acessível, mas que está sujeito a contaminação microbiológica, acarretando perigo potencial aos consumidores, principalmente se Salmonella estiver presente. Fungos também representam sério problema para a qualidade de ovos, podendo levar à rejeição pelo consumidor. Visando solucionar este problema, desenvolveu-se um revestimento a base de isolado proteico de soro de leite (IPS) com nanopartículas de montmorilonita adicionado do agente antimicrobiano metabissulfito de sódio. A pesquisa objetivou avaliar a atividade antimicrobiana do revestimento contra Salmonella Enteritidis, Aspergillus sydowii e Penicillium paxilli quando aplicado em ovos brancos. Não houve diferença significativa (p > 0,05) entre as populações de S. Enteritidis das cascas revestidas e sem revestimento. A. sydowii e P. paxilli apresentaram crescimento tanto nas cascas de ovos sem revestimento quanto nas cascas de ovos revestidos, evidenciando que o revestimento não foi capaz de inibi-los nas condições testadas. Conclui-se que o revestimento desenvolvido não apresenta atividade antimicrobiana contra S. Enteritidis, A. sydowii e P. paxilli nas condições testadas.

Regiospecificities and Prenylation Mode Specificities of the Fungal Indole Diterpene Prenyltransferases AtmD and PaxD

ABSTRACTWe recently reported the function ofpaxD, which is involved in the paxilline (compound 1) biosynthetic gene cluster inPenicillium paxilli. Recombinant PaxD catalyzed a stepwise regular-type diprenylation at the 21 and 22 positions of compound 1 with dimethylallyl diphosphate (DMAPP) as the prenyl donor. In this study,atmD, which is located in the aflatrem (compound 2) biosynthetic gene cluster inAspergillus flavusand encodes an enzyme with 32% amino acid identity to PaxD, was characterized using recombinant enzyme. When compound 1 and DMAPP were used as substrates, two major products and a trace of minor product were formed. The structures of the two major products were determined to be reversely monoprenylated compound 1 at either the 20 or 21 position. Because compound 2 and β-aflatrem (compound 3), both of which are compound 1-related compounds produced byA. flavus, have the same prenyl moiety at the 20 and 21 position, respectively, AtmD should catalyze the prenylation in compound 2 and 3 biosynthesis. More importantly and surprisingly, AtmD accepted paspaline (compound 4), which is an intermediate of compound 1 biosynthesis that has a structure similar to that of compound 1, and catalyzed a regular monoprenylation of compound 4 at either the 21 or 22 position, though the reverse prenylation was observed with compound 1. This suggests that fungal indole diterpene prenyltransferases have the potential to alter their position and regular/reverse specificities for prenylation and could be applicable for the synthesis of industrially useful compounds.

Identification of Two Aflatrem Biosynthesis Gene Loci in Aspergillus flavus and Metabolic Engineering of Penicillium paxilli To Elucidate Their Function

ABSTRACT Aflatrem is a potent tremorgenic toxin produced by the soil fungus Aspergillus flavus, and a member of a structurally diverse group of fungal secondary metabolites known as indole-diterpenes. Gene clusters for indole-diterpene biosynthesis have recently been described in several species of filamentous fungi. A search of Aspergillus complete genome sequence data identified putative aflatrem gene clusters in the genomes of A. flavus and Aspergillus oryzae. In both species the genes for aflatrem biosynthesis cluster at two discrete loci; the first, ATM1, is telomere proximal on chromosome 5 and contains a cluster of three genes, atmG, atmC, and atmM, and the second, ATM2, is telomere distal on chromosome 7 and contains five genes, atmD, atmQ, atmB, atmA, and atmP. Reverse transcriptase PCR in A. flavus demonstrated that aflatrem biosynthesis transcript levels increased with the onset of aflatrem production. Transfer of atmP and atmQ into Penicillium paxilli paxP and paxQ deletion mutants, known to accumulate paxilline intermediates paspaline and 13-desoxypaxilline, respectively, showed that AtmP is a functional homolog of PaxP and that AtmQ utilizes 13-desoxypaxilline as a substrate to synthesize aflatrem pathway-specific intermediates, paspalicine and paspalinine. We propose a scheme for aflatrem biosynthesis in A. flavus based on these reconstitution experiments in P. paxilli and identification of putative intermediates in wild-type cultures of A. flavus.