Mary N. Heneghan

Authentic Heterologous Expression of the Tenellin Iterative Polyketide Synthase Nonribosomal Peptide Synthetase Requires Coexpression with an Enoyl Reductase

The tenS gene encoding tenellin synthetase (TENS), a 4239‐residue polyketide synthase nonribosomal‐peptide synthetase (PKS‐NRPS) from Beauveria bassiana, was expressed in Aspergillus oryzae M‐2‐3. This led to the production of three new compounds, identified as acyl tetramic acids, and numerous minor metabolites. Consideration of the structures of these compounds indicates that the putative C‐terminal thiolester reductase (R) domain does not act as a reductase, but appears to act as a Dieckmann cyclase (DKC). Expression of tenS in the absence of a trans‐acting ER component encoded by orf3 led to errors in assembly of the polyketide component, giving clues to the mode of programming of highly reducing fungal PKS. Coexpression of tenS with orf3 from the linked gene cluster led to the production of a correctly elaborated polyketide. The NRPS adenylation domain possibly shows the first identified fungal signature sequences for tyrosine selectivity.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the entomopathogenic fungus Beauveria bassiana.

Late stage oxidations during the biosynthesis of the 2-pyridone tenellin in the insect pathogenic fungus Beauveria bassiana were investigated by a combination of gene knockout, antisense RNA, and gene coexpression studies. Open reading frames (ORF) 3 and 4 of the tenellin biosynthetic gene cluster were previously shown to encode a trans-acting enoyl reductase and a hybrid polyketide synthase nonribosomal peptide synthetase (PKS-NRPS), respectively, which together synthesize the acyltetramic acid pretenellin-A. In this work, we have shown that ORF1 encodes a cytochrome P450 oxidase, which catalyzes an unprecedented oxidative ring expansion of pretenellin-A to form the 2-pyridone core of tenellin and related metabolites, and that this enzyme does not catalyze the formation of a hydroxylated precursor. Similar genes appear to be associated with PKS-NRPS genes in other fungi. ORF2 encodes an unusual cytochrome P450 monooxygenase required for the selective N-hydroxylation of the 2-pyridone which is incapable of N-hydroxylation of acyltetramic acids.

First Heterologous Reconstruction of a Complete Functional Fungal Biosynthetic Multigene Cluster

Fungal natural products include antibiotics such as the penicillins, antirejection drugs such as cyclosporins and cholesterollowering drugs such as the statins. High productivity of these pharmaceutically important compounds is desirable and has regularly been pursued by strain improvement and metabolic engineering. Recently partial and full genome sequencing has revealed clusters of genes encoding the biosynthesis of these compounds in fungi. Surprisingly it has been found that while rich in bioactive compounds, fungi are even richer in biosynthetic gene clusters. For example, in the well-characterised species Aspergillus nidulans, there are at least 54 secondary metabolite gene clusters, only half of which are characterised at a chemical level. Fungal secondary metabolite gene clusters can be readily identified from key genes within the clusters, such as nonribosomal peptide synthetases (NRPS), terpene cyclases or polyketide synthases (PKS). Although it is relatively easy to identify such gene clusters, it is currently almost impossible to predict the chemical product synthesised, as the programming of the synthase cannot be predicted from sequence information alone. This is further compounded by the presence of genes for many tailoring enzymes, which usually act after the core synthase has completed synthesis of the skeleton, further modifying the resulting compound, making it extremely difficult to elucidate the likely metabolite from gene sequence data alone. A number of different strategies have been employed to decipher fungal gene clusters. These include: chemical profiling of cultures produced under a range of different conditions; over-expression of pathway regulatory genes (where present) ; manipulation of transcriptional activators (e.g. , LaeA) ; manipulation of the pH regulatory system (e.g. , PacC) ; use of chromatin modifying (i.e. , epigenetic) chemicals ; and cofermentation of bacteria and fungi. Unfortunately all of these techniques are restricted in their general utility. Many of the biosynthetic clusters do not include specific transcriptional regulators, many pathways do not respond to LaeA or PacC regulation and chromatin modifiers only activate a small subset of the gene clusters within a genome. In addition, many fungi are difficult to cultivate on a large scale and are often recalcitrant to molecular techniques, so these approaches cannot be applied. We and others have used another approach to investigate the biosynthesis of biologically active compounds in fungi that of heterologous gene expression. This method involves transfer of a gene of interest from a donor strain to a suitable host. In principle a bacterial host such as E. coli could be used, but several problems are usually encountered. First, bacterial hosts are unable to process eukaryotic introns and so these must be removed. Second, expression of eukaryotic genes in bacteria can be problematic, especially if there is a significant codon bias. Third, bacteria can experience difficulty in correctly folding fungal polypeptides. Fourth, proteins such as PKS and NRPS require selective post-translational phosphopantetheinylation for them to be active in vivo. Finally, bacteria may not supply specific metabolites for biosynthesis. While each of these problems can be overcome in isolation, cumulative effects can make the use of bacteria as expression hosts for fungal genes troublesome and inefficient. This is illustrated by the recent expression of the beauvericin NRPS (bbBeas) from

The programming role of trans-acting enoyl reductases during the biosynthesis of highly reduced fungal polyketides

A novel polyketide synthase nonribosomal peptide synthetase (PKS-NRPS) genecluster was isolated from Beauveria bassiania 992.05. The cluster encodes the enzymes responsible for the biosynthesis of the new 2-pyridone desmethylbassianin (DMB). DMB is structurally related to tenellin from B. bassiana 110.25 but it differs in chain length and degree of methylation. Despite these programming differences the 20 kb DMB biosynthetic genecluster has 90% sequence identity to the tenellingenecluster. Silencing of the PKS-NRPS gene, dmbS, resulted in total loss of DMB production. Co-expression of dmbS in Aspergillus oryzae with its cognate trans-actingenoyl reductase gene, dmbC, produced predesmethylbassianin A, the first isolable precursor in the biosynthetic pathway. Expression of dmbS with the tenellintrans-actingenoyl reductase gene, tenC, also resulted in the production of predesmethylbassianin A. Co-expression of tenS, the tenellin PKS-NRPS, with dmbC produced pretenellin A. These results show that the tenS and dmbS encoded PKS-NRPS contains the programme for polyketide biosynthesis, while the trans-actingERs appear to control the fidelity of the programme. Expression of a hybrid synthetase in which the PKS of the tenellin synthetase was fused to the NRPS from DMBS produced prototenellins A to C, indicating that the NRPS does not act as a selecting gatekeeper to affect the PKS programme.

A comparison of methods for successful triggering of gene silencing in Coprinus cinereus

Post-transcriptional gene-silencing methods (PTGS), including RNAi, are becoming increasingly pervasive in functional genomics. To advance analysis of the recently sequenced Coprinus cinereus genome, a high throughput gene silencing method is essential. We have exploited the GFP reporter gene to evaluate and quantify efficacy of three different silencing strategies. Modular constructs that encompassed antisense, untranslatable sense, and RNAi-mediating hairpin sequences, were transformed into a GFP-expressing host strain. Transformants exhibiting strong downregulation and partial suppression of GFP were recovered with all three constructs. Analyses of protein and transcriptional nucleic acids revealed that the antisense and hairpin sequences yielded similar levels of GFP suppression, and were both more efficient than untranslatable sense sequences. Our antisense vectors will expedite functional characterisation of C. cinereus and the modular nature of the constructs should permit exploitation of directional cDNA libraries for high throughput screening.

Characterization of Serine Proteinase Expression in Agaricus bisporus and Coprinopsis cinerea by Using Green Fluorescent Protein and the A. bisporus SPR1 Promoter

ABSTRACT The Agaricus bisporus serine proteinase 1 (SPR1) appears to be significant in both mycelial nutrition and senescence of the fruiting body. We report on the construction of an SPR promoter::green fluorescent protein (GFP) fusion cassette, pGreen_hph1_SPR_GFP, for the investigation of temporal and developmental expression of SPR1 in homobasidiomycetes and to determine how expression is linked to physiological and environmental stimuli. Monitoring of A. bisporus pGreen_hph1_SPR_GFP transformants on media rich in ammonia or containing different nitrogen sources demonstrated that SPR1 is produced in response to available nitrogen. In A. bisporus fruiting bodies, GFP activity was localized to the stipe of postharvest senescing sporophores. pGreen_hph1_SPR_GFP was also transformed into the model basidiomycete Coprinopsis cinerea. Endogenous C. cinerea proteinase activity was profiled during liquid culture and fruiting body development. Maximum activity was observed in the mature cap, while activity dropped during autolysis. Analysis of the C. cinerea genome revealed seven genes showing significant homology to the A. bisporus SPR1 and SPR2 genes. These genes contain the aspartic acid, histidine, and serine residues common to serine proteinases. Analysis of the promoter regions revealed at least one CreA and several AreA regulatory motifs in all sequences. Fruiting was induced in C. cinerea dikaryons, and fluorescence was determined in different developmental stages. GFP expression was observed throughout the life cycle, demonstrating that serine proteinase can be active in all stages of C. cinerea fruiting body development. Serine proteinase expression (GFP fluorescence) was most concentrated during development of young tissue, which may be indicative of high protein turnover during cell differentiation.

Functional analysis of Agaricus bisporus serine proteinase 1 reveals roles in utilization of humic rich substrates and adaptation to the leaf-litter ecological niche

Summary Agaricus bisporus is a secondary decomposer fungus and an excellent model for the adaptation, persistence and growth of fungi in humic‐rich environments such as soils of temperate woodland and pastures. The A. bisporus serine proteinase SPR1 is induced by humic acids and is highly expressed during growth on compost. Three Spr1 gene silencing cassettes were constructed around sense, antisense and non‐translatable‐stop strategies (pGRsensehph, pGRantihph and pGRstophph). Transformation of A. bisporus with these cassettes generated cultures showing a reduction in extracellular proteinase activity as demonstrated by the reduction, or abolition, of a clearing zone on plate‐based bioassays. These lines were then assessed by detailed enzyme assay, RT‐qPCR and fruiting. Serine proteinase activity in liquid cultures was reduced in 83% of transformants. RT‐qPCR showed reduced Spr1 mRNA levels in all transformants analysed, and these correlated with reduced enzyme activity. When fruiting was induced, highly‐silenced transformant AS5 failed to colonize the compost, whilst for those that did colonize the compost, 60% gave a reduction in mushroom yield. Transcriptional, biochemical and developmental observations, demonstrate that SPR1 has an important role in nutrient acquisition in compost and that SPR1 is a key enzyme in the adaptation of Agaricus to the humic‐rich ecological niche formed during biomass degradation.