Visualizing Metabolism in Biotechnologically Important Yeasts with dDNP NMR Reveals Evolutionary Strategies and Glycolytic Logic.

Saccharomyces cerevisiae has long been a pillar of biotechnological production and basic research. More recently, strides to exploit the functional repertoire of nonconventional yeasts for biotechnological production have been made. Genomes and genetic tools for these yeasts are not always available, and yeast genomics alone may be insufficient to determine the functional features in yeast metabolism. Hence, functional assays of metabolism, ideally in the living cell, are best suited to characterize the cellular biochemistry of such yeasts. Advanced in cell NMR methods can allow the direct observation of carbohydrate influx into central metabolism on a seconds time scale: dDNP NMR spectroscopy temporarily enhances the nuclear spin polarization of substrates by more than 4 orders of magnitude prior to functional assays probing central metabolism. We use various dDNP enhanced carbohydrates for in-cell NMR to compare the metabolism of S. cerevisiae and nonconventional yeasts, with an emphasis on the wine yeast Hanseniaspora uvarum. In-cell observations indicated more rapid exhaustion of free cytosolic NAD+ in H. uvarum and alternative routes for pyruvate conversion, in particular, rapid amination to alanine. In-cell observations indicated that S. cerevisiae outcompetes other biotechnologically relevant yeasts by rapid ethanol formation due to the efficient adaptation of cofactor pools and the removal of competing reactions from the cytosol. By contrast, other yeasts were better poised to use redox neutral processes that avoided CO2-emission. Beyond visualizing the different cellular strategies for arriving at redox neutral end points, in-cell dDNP NMR probing showed that glycolytic logic is more conserved: nontoxic precursors of cellular building blocks formed high-population intermediates in the influx of glucose into the central metabolism of eight different biotechnologically important yeasts. Unsupervised clustering validated that the observation of rapid intracellular chemistry is a viable means to functionally classify biotechnologically important organisms.

Glycoengineering of Aspergillus nidulans to produce precursors for humanized N-glycan structures.

Filamentous fungi secrete protein with a very high efficiency, and this potential can be exploited advantageously to produce therapeutic proteins at low costs. A significant barrier to this goal is posed by the fact that fungal N-glycosylation varies substantially from that of humans. Inappropriate N-glycosylation of therapeutics results in reduced product quality, including poor efficacy, decreased serum half-life, and undesirable immune reactions. One solution to this problem is to reprogram the glycosylation pathway of filamentous fungi to decorate proteins with glycans that match, or can be remodeled into, those that are accepted by humans. In yeast, deletion of ALG3 leads to the accumulation of Man5GlcNAc2 glycan structures that can act as a precursor for remodeling. However, in Aspergilli, deletion of the ALG3 homolog algC leads to an N-glycan pool where the majority of the structures contain more hexose residues than the Man3-5GlcNAc2 species that can serve as substrates for humanized glycan structures. Hence, additional strain optimization is required. In this report, we have used gene deletions in combination with enzymatic and chemical glycan treatments to investigate N-glycosylation in the model fungus Aspergillus nidulans. In vitro analyses showed that only some of the N-glycan structures produced by a mutant A. nidulans strain, which is devoid of any of the known ER mannose transferases, can be trimmed into desirable Man3GlcNAc2 glycan structures, as substantial amounts of glycan structures appear to be capped by glucose residues. In agreement with this view, deletion of the ALG6 homolog algF, which encodes the putative α-1,3- glucosyltransferase that adds the first glucose residue to the growing ER glycan structure, dramatically reduces the amounts of Hex6-7HexNAc2 structures. Similarly, these structures are also sensitive to overexpression of the genes encoding the heterodimeric α-glucosidase II complex. Without the glucose caps, a new set of large N-glycan structures was formed. Formation of this set is mostly, perhaps entirely, due to mannosylation, as overexpression of the gene encoding mannosidase activity led to their elimination. Based on our new insights into the N-glycan processing in A. nidulans, an A. nidulans mutant strain was constructed in which more than 70% of the glycoforms appear to be Man3-5GlcNAc2 species, which may serve as precursors for further engineering in order to create more complex human-like N-glycan structures.

Loss of AA13 LPMOs impairs degradation of resistant starch and reduces the growth of Aspergillus nidulans.

BACKGROUND: Lytic polysaccharide monooxygenases (LPMOs) are often studied in simple models involving activity measurements of a single LPMO or a blend thereof with hydrolytic enzymes towards an insoluble substrate. However, the contribution of LPMOs to polysaccharide breakdown in complex cocktails of hydrolytic and oxidative enzymes, similar to fungal secretomes, remains elusive. Typically, two starch-specific AA13 LPMOs are encoded by mainly Ascomycota genomes. Here, we investigate the impact of LPMO loss on the growth and degradation of starches of varying resistance to amylolytic hydrolases by Aspergillus nidulans. RESULTS: Deletion of the genes encoding AnAA13A that possesses a CBM20 starch-binding module, AnAA13B (lacking a CBM20) or both AA13 genes resulted in reduced growth on solid media with resistant, but not soluble processed potato starch. Larger size and amount of residual starch granules were observed for the AA13-deficient strains as compared to the reference and the impairment of starch degradation was more severe for the strain lacking AnAA13A based on a microscopic analysis. After 5 days of growth on raw potato starch in liquid media, the mount of residual starch was about fivefold higher for the AA13 gene deletion strains compared to the reference, which underscores the importance of LPMOs for degradation of especially resistant starches. Proteomic analyses revealed substantial changes in the secretomes of the double AA13 gene deletion, followed by the AnAA13A-deficient strain, whereas only a single protein was significantly different in the proteome of the AnAA13B-deficient strain as compared to the reference. CONCLUSIONS: This study shows that the loss of AA13, especially the starch-binding AnAA13A, impairs degradation of resistant potato starch, but has limited impact on less-resistant wheat starch and no impact on processed solubilized starch. The effects of LPMO loss are more pronounced at the later stages of fungal growth, likely due to the accumulation of the less-accessible regions of the substrate. The striking impairment in granular starch degradation due to the loss of a single LPMO from the secretome offers insight into the crucial role played by AA13 in the breakdown of resistant starch and presents a methodological framework to analyse the contribution of distinct LPMOs towards semi-crystalline polysaccharides under in vivo conditions.

Uncovering secondary metabolite evolution and biosynthesis using gene cluster networks and genetic dereplication.

The increased interest in secondary metabolites (SMs) has driven a number of genome sequencing projects to elucidate their biosynthetic pathways. As a result, studies revealed that the number of secondary metabolite gene clusters (SMGCs) greatly outnumbers detected compounds, challenging current methods to dereplicate and categorize this amount of gene clusters on a larger scale. Here, we present an automated workflow for the genetic dereplication and analysis of secondary metabolism genes in fungi. Focusing on the secondary metabolite rich genus Aspergillus, we categorize SMGCs across genomes into SMGC families using network analysis. Our method elucidates the diversity and dynamics of secondary metabolism in section Nigri, showing that SMGC diversity within the section has the same magnitude as within the genus. Using our genome analysis we were able to predict the gene cluster responsible for biosynthesis of malformin, a potentiator of anti-cancer drugs, in 18 strains. To proof the general validity of our predictions, we developed genetic engineering tools in Aspergillus brasiliensis and subsequently verified the genes for biosynthesis of malformin.

Genes Linked to Production of Secondary Metabolites in Talaromyces atroroseus Revealed Using CRISPR-Cas9.

The full potential of fungal secondary metabolism has until recently been impeded by the lack of universal genetic tools for most species. However, the emergence of several CRISPR-Cas9-based genome editing systems adapted for several genera of filamentous fungi have now opened the doors for future efforts in discovery of novel natural products and elucidation and engineering of their biosynthetic pathways in fungi where no genetic tools are in place. So far, most studies have focused on demonstrating the performance of CRISPR-Cas9 in various fungal model species, and recently we presented a versatile CRISPR-Cas9 system that can be successfully applied in several diverse Aspergillus species. Here we take it one step further and show that our system can be used also in a phylogenetically distinct and largely unexplored species from the genus of Talaromyces. Specifically, we exploit CRISPR-Cas9-based genome editing to identify a new gene in T. atroroseus responsible for production of polyketide-nonribosomal peptide hybrid products, hence, linking fungal secondary metabolites to their genetic origin in a species where no genetic engineering has previously been performed.

Effect of secretory pathway gene overexpression on secretion of a fluorescent reporter protein in Aspergillus nidulans.

BACKGROUND: The considerable capacity of filamentous fungi for the secretion of proteins is the basis for multi-billion dollar industries producing enzymes and proteins with therapeutic value. The stepwise pathway from translation to secretion is therefore well studied, and genes playing major roles in the process have been identified through transcriptomics. The assignment of function to these genes has been enabled in combination with gene deletion studies. In this work, 14 genes known to play a role in protein secretion in filamentous fungi were overexpressed in Aspergillus nidulans. The background strain was a fluorescent reporter secreting mRFP. The overall effect of the overexpressions could thus be easily monitored through fluorescence measurements, while the effects on physiology were determined in batch cultivations and surface growth studies. RESULTS: Fourteen protein secretion pathway related genes were overexpressed with a tet-ON promoter in the RFP-secreting reporter strain and macromorphology, physiology and protein secretion were monitored when the secretory genes were induced. Overexpression of several of the chosen genes was shown to cause anomalies on growth, micro- and macro-morphology and protein secretion levels. While several constructs exhibited decreased secretion of the model protein, the overexpression of the Rab GTPase RabD resulted in a 40 % increase in secretion in controlled bioreactor cultivations. Fluorescence microscopy revealed alterations of protein localization in some of the constructed strains, giving further insight into potential roles of the investigated genes. CONCLUSIONS: This study demonstrates the possibility of significantly increasing cellular recombinant protein secretion by targeted overexpression of secretion pathway genes. Some gene targets investigated here, including genes from different compartments of the secretory pathway resulted in no significant change in protein secretion, or in significantly lowered protein titres. As the 14 genes selected in this study were previously shown to be upregulated during protein secretion, our results indicate that increased expression may be a way for the cell to slow down secretion in order to cope with the increased protein load. By constructing a secretion reporter strain, the study demonstrates a robust way to study the secretion pathway in filamentous fungi.

Linker Flexibility Facilitates Module Exchange in Fungal Hybrid PKS-NRPS Engineering.

Polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) each give rise to a vast array of complex bioactive molecules with further complexity added by the existence of natural PKS-NRPS fusions. Rational genetic engineering for the production of natural product derivatives is desirable for the purpose of incorporating new functionalities into pre-existing molecules, or for optimization of known bioactivities. We sought to expand the range of natural product diversity by combining modules of PKS-NRPS hybrids from different hosts, hereby producing novel synthetic natural products. We succeeded in the construction of a functional cross-species chimeric PKS-NRPS expressed in Aspergillus nidulans. Module swapping of the two PKS-NRPS natural hybrids CcsA from Aspergillus clavatus involved in the biosynthesis of cytochalasin E and related Syn2 from rice plant pathogen Magnaporthe oryzae lead to production of novel hybrid products, demonstrating that the rational re-design of these fungal natural product enzymes is feasible. We also report the structure of four novel pseudo pre-cytochalasin intermediates, niduclavin and niduporthin along with the chimeric compounds niduchimaeralin A and B, all indicating that PKS-NRPS activity alone is insufficient for proper assembly of the cytochalasin core structure. Future success in the field of biocombinatorial synthesis of hybrid polyketide-nonribosomal peptides relies on the understanding of the fundamental mechanisms of inter-modular polyketide chain transfer. Therefore, we expressed several PKS-NRPS linker-modified variants. Intriguingly, the linker anatomy is less complex than expected, as these variants displayed great tolerance with regards to content and length, showing a hitherto unreported flexibility in PKS-NRPS hybrids, with great potential for synthetic biology-driven biocombinatorial chemistry.

Diversity in Secondary Metabolites Including Mycotoxins from Strains of Aspergillus Section Nigri Isolated from Raw Cashew Nuts from Benin, West Africa.

In a previous study, raw cashew kernels were assayed for the fungal contamination focusing on strains belonging to the genus Aspergillus and on aflatoxins producers. These samples showed high contamination with Aspergillus section Nigri species and absence of aflatoxins. To investigate the diversity of secondary metabolites, including mycotoxins, the species of A. section Nigri may produce and thus threaten to contaminate the raw cashew kernels, 150 strains were isolated from cashew samples and assayed for their production of secondary metabolites using liquid chromatography high resolution mass spectrometry (LC-HRMS). Seven species of black Aspergilli were isolated based on morphological and chemical identification: A. tubingensis (44%), A. niger (32%), A. brasiliensis (10%), A. carbonarius (8.7%), A. luchuensis (2.7%), A. aculeatus (2%) and A. aculeatinus (0.7%). From these, 45 metabolites and their isomers were identified. Aurasperone and pyranonigrin A, produced by all species excluding A. aculeatus and A. aculeatinus, were most prevalent and were encountered in 146 (97.3%) and 145 (95.7%) isolates, respectively. Three mycotoxins groups were detected: fumonisins (B2 and B4) (2.7%) ochratoxin A (13.3%), and secalonic acids (2%), indicating that these mycotoxins could occur in raw cashew nuts. Thirty strains of black Aspergilli were randomly sampled for verification of species identity based on sequences of ß-tubulin and calmodulin genes. Among them, 27 isolates were positive to the primers used and 11 were identified as A. niger, 7 as A. tubingensis, 6 as A. carbonarius, 2 as A. luchuensis and 1 as A. welwitschiae confirming the species names as based on morphology and chemical features. These strains clustered in 5 clades in A. section Nigri. Chemical profile clustering also showed also 5 groups confirming the species specific metabolites production.