Current state of genome-scale modeling in filamentous fungi

The group of filamentous fungi contains important species used in industrial biotechnology for acid, antibiotics and enzyme production. Their unique lifestyle turns these organisms into a valuable genetic reservoir of new natural products and biomass degrading enzymes that has not been used to full capacity. One of the major bottlenecks in the development of new strains into viable industrial hosts is the alteration of the metabolism towards optimal production. Genome-scale models promise a reduction in the time needed for metabolic engineering by predicting the most potent targets in silico before testing them in vivo. The increasing availability of high quality models and molecular biological tools for manipulating filamentous fungi renders the model-guided engineering of these fungal factories possible with comprehensive metabolic networks. A typical fungal model contains on average 1138 unique metabolic reactions and 1050 ORFs, making them a vast knowledge-base of fungal metabolism. In the present review we focus on the current state as well as potential future applications of genome-scale models in filamentous fungi

A brief journey into the history of, and future sources and uses of fatty acids

The authors would like to thank the Engineering and Physical Sciences Research Council, University of St. Andrews, and the EPSRC Centre for Doctoral Training in Critical Resource Catalysis (CRITICAT) for financial support (Ph.D. studentship to MC; Grant code: EP/L016419/1).Fats and lipids have always had a primarily role in the history of humankind, from the ancient civilisations to the modern and contemporary time, going from domestic and cosmetic uses, to the first medical applications and later to the large scale industrial uses for food, pharmaceutical, cosmetics and biofuel production. Sources and uses of those have changed during time following the development of chemical sciences and industrial technological advances. Plants, fish and animal fats have represented the primary source of lipids and fats for century. Nowadays the use of fatty acid sources has taken a turn: industries are mainly interested in polyunsaturated fatty acids (PUFAs), which have beneficial properties in human health; and also, for high-value fatty acids product for innovative and green production of biofuel and feedstocks. Thus, the constant increase in demand of fatty acids, the fact that marine and vegetable sources are not adequate to meet the high level of fatty acids required worldwide and climate change, have determined the necessity of the search for renewable and sustainable sources for fatty acids. Biotechnological advances and bioengineering have started looking at the genetic modification of algae, bacteria, yeasts, seeds and plants to develop cell-factory able to produce high value fatty acid products in renewable and sustainable manner. This innovative approach applied to FAs industry is a peculiar example of how biotechnology can serve as powerful mean to drive the production of high value fatty acid derivatives on the concept of circular bioeconomy, based on the reutilisation of organic resources for alternative and sustainable productive patterns that are environmentally friendly.Publisher PDFPeer reviewe

Reconstruction and Analysis of a Genome-Scale Metabolic Model of Ganoderma lucidum for Improved Extracellular Polysaccharide Production

In this study, we reconstructed for the first time a genome-scale metabolic model (GSMM) of Ganoderma lucidum strain CGMCC5.26, termed model iZBM1060, containing 1060 genes, 1202 metabolites, and 1404 reactions. Important findings based on model iZBM1060 and its predictions are as follows: (i) The extracellular polysaccharide (EPS) biosynthetic pathway was elucidated completely. (ii) A new fermentation strategy is proposed: addition of phenylalanine increased EPS production by 32.80% in simulations and by 38.00% in experiments. (iii) Eight genes for key enzymes were proposed for EPS overproduction. Model iZBM1060 provides a useful platform for regulating EPS production in terms of system metabolic engineering for G. lucidum, as well as a guide for future metabolic pathway construction of other high value-added edible/ medicinal mushroom species

Biosynthese und Funktion von Naturstoffen aus Mortierella alpina

In dieser Arbeit wurde anhand des Modellorganismus Mortierella alpina gezeigt, dass basale Pilze wohl zu ausgedehnter Naturstoffbiosyntese befähigt sind. So konnte für die Biosynthese der Malpinine, der Malpicycline, der Malpibaldine sowie das Calpinactam jeweils eine zugehörige Nicht-ribosomale Peptidsynthetase (NRPSen) identifziert werden. Bei den jeweils zugrundeliegenden Biosynthesegenen malA, mpbA, calA und mpcA handelt es sich um pilzliche Gene, die jedoch bakteriellen Ursprungs sind und durch horizontalen Gentransfer auf M. alpina übertragen worden sind. Zusätzlich wurde die biologische Funktion sowie mögliche pharmazeutische Anwendungsmöglichkeiten untersucht

Metabolic and regulatory network analysis of DHA-producing recombinant Yarrowia lipolytica

Heterologous expression of myxobacterial PKS-like PUFA cluster in recently created recombinant Yarrowia lipolytica Af4 strain allowed formation of DHA, polyunsaturated fatty acid with significant impact on human health. The focus of this work was to unravel the complex underlying metabolism of the oleaginous yeast using systems biology tools. Growth-decoupled production of DHA occurred during the stationary phase. The transition triggered drastic changes in global transcription and reduced availability of the two DHA precursors, acetyl and malonyl-CoA up to 98%. Upregulation of genes belonging to PUFA cluster coincided with the activated degradation of lipid bodies and fatty acids and the associated glyoxylate shunt, reactions involved in cellular stress-response, and interestingly, catabolic routes of branched-chain amino acids and L-lysine. This inspired the creation of time-resolved feeding strategies that supplemented the cultures with citrate, L-lysine, L-leucine and L-isoleucine, respectively to provide additional acetyl-CoA. Beneficially, the novel strategy increased DHA production up to 38%. Careful 13C tracer studies then unraveled that the supplemented carbon was significantly incorporated into intracellular CoA-esters, amino acids, and DHA itself. Fed by small amounts of L-lysine, the recombinant producer Y. lipolytica Af4 accumulated DHA up to the gram scale in the fed-batch process, and surpassed previous efforts three-fold.Die heterologe Expression eines myxobakteriellen PUFA-Clusters in dem kürzlich geschaffenen rekombinanten Yarrowia lipolytica Af4 ermöglichte die Synthese von DHA, einer mehrfach ungesättigten Fettsäure mit erheblichen Auswirkungen auf die Gesundheit. Der Schwerpunkt dieser Arbeit lag auf der Aufklärung des komplexen zugrunde liegenden Stoffwechsels der öligen Hefe mit systembiologischen Methoden. Die Produktion von DHA erfolgte wachstumsentkoppelt in der stationären Phase. Der Übergang löste drastische Veränderungen in der globalen Transkription aus und verringerte die Verfügbarkeit der beiden DHA-Vorstufen Acetyl- und Malonyl-CoA um bis zu 98%. Die Hochregulierung des PUFA-Clusters, fiel mit dem aktivierten Abbau von Lipiden und dem damit verbundenen Glyoxylat-Shunt, mit verstärkter Stressreaktion, und interessanterweise auch mit katabolen Wegen von verzweigtkettigen Aminosäuren und L-Lysin zusammen. Dies inspirierte zur Entwicklung von zeitaufgelösten Fütterungsstrategien, bei denen Citrat, L Lysin, L Leucin bzw. L Isoleucin zugeführt wurden, um zusätzliches Acetyl-CoA bereitzustellen. Mit dieser neuen Strategie konnte die DHA-Produktion um bis zu 38% gesteigert werden. 13C-Tracer-Studien ergaben, dass der zugeführte Kohlenstoff in großem Umfang in CoA-Ester, Aminosäuren und DHA eingebaut wurde. Im Fed-Batch-Verfahren produzierte Y. lipolytica Af4, der mit geringen Mengen L Lysin gefüttert wurde, DHA bis in den Grammbereich und übertraf frühere Ansätze um das Dreifache

Fungi and Fungal Metabolites for the Improvement of Human and Animal Nutrition and Health

The purpose of this book was not to provide a comprehensive overview of the vast arena of how fungi and fungal metabolites are able to improve human and animal nutrition and health; rather, we, as Guest Editors, wished to encourage authors working in this field to publish their most recent work in this rapidly growing journal in order for the large readership to appreciate the full potential of wonderful and beneficial fungi. Thus, this Special Issue welcomed scientific contributions on applications of fungi and fungal metabolites, such as bioactive fatty acids, pigments, polysaccharides, alkaloids, terpenoids, etc., with great potential in human and animal nutrition and health

Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger

Background: Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. Results: We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the\ua0Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. Conclusions: This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.[Figure not available: see fulltext.]

Genomic insights into the lifestyles, functional capacities and oleagenicity of members of the fungal family Trichosporonaceae

Trichosporonaceae incorporates six genera of physiologically and ecologically diverse fungi including both human pathogenic taxa as well as yeasts of biotechnological interest, especially those oleagenic taxa that accumulate large amounts of single cell oils (SCOs). Here, we have undertaken comparative genomic analysis of thirty-three members of the family with a view to gain insight into the molecular determinants underlying their lifestyles and niche specializations. Phylogenomic analysis revealed potential misidentification of three strains which could impact subsequent analyses. Evaluation of the predicted proteins coding sequences showed that the free-living members of the family harbour greater numbers of carbohydrate active enzymes (CAZYmes), metallo- and serine peptidases compared to their host-associated counterparts. Phylogenies of selected lipid biosynthetic enzymes encoded in the genomes of the studied strains revealed disparate evolutionary histories for some proteins inconsistent with the core genome phylogeny. However, the documented oleagenic members distinctly cluster based on the constitution of the upstream regulatory regions of genes encoding acetyl-CoA carboxylase (ACC), ATP-citrate synthase (ACS) and isocitrate dehydrogenase [NADP] (ICDH), which are among the major proteins in the lipid biosynthetic pathway of these yeasts, suggesting a possible pattern in the regulation of these genes

The modification of plant oil composition via metabolic engineering - better nutrition by design

This article will focus on the modification of plant seed oils to enhance their nutritional composition. Such modifications will include C18 ?6-desaturated fatty acids such as ?-linolenic and stearidonic acid, omega-6 long-chain polyunsaturated fatty acids such as arachidonic acid, as well as the omega-3 long-chain polyunsaturated fatty acids (often named fish oils) such as eicosapentaenoic acid and docosahexaenoic acid. We will consider how new technologies (such as synthetic biology, next-generation sequencing and lipidomics) can help speed up and direct the development of desired traits in transgenic oilseeds. We will also discuss how manipulating triacylglycerol structure can further enhance the nutritional value of designer oils. We will also consider how advances in model systems have translated into crops and the potential end-users for such novel oils (e.g. aquaculture, animal feed, human nutrition)