Mycology: Current and Future Developments

In 2013, the world production of ethanol was about 23.4 billion gallons. However, because of a global increase in fuel consumption, an increase in bioethanol production is necessary. The search for new energy sources increased the attention on biomass; now it is used directly for energy cogeneration by combustion and for the production of new fuels such as cellulosic ethanol or lignocellulosic ethanol, also called second-generation (2G) ethanol. Bioethanol production employing renewable sources is increasingly in demand worldwide because of the continuous depletion of fossil fuels, economic and political crises, and growing environmental safety concerns. Brazil and USA are the two largest producers and exporters of ethanol in the world. Nevertheless, other countries including China, India, Canada, Japan, Colombia, and Argentina have assumed featured positions in global fuel ethanol production. Therefore, this new world order may result in the development of an industrially suitable production strategy that will solve our energy crisis by producing more ethanol sustainably.

Currently, bioethanol is the most promising alternative renewable energy source to fossil fuels. Bioethanol has the potential to provide sustainable, cost effective energy while reducing greenhouse gas emissions. The complex lignin-cellulosehemicellulose matrix of the biomass has to be fractioned and the carbohydrate polymers need to undergo hydrolysis to yield fermentable sugars. This hydrolysis step is typically catalyzed by cocktails of enzymes including cellulases, hemicellulases and other accessory proteins that target and degrade specific constituents of cell wall. The requirements of enzyme complexes which act synergistically to unlock and saccharify polysaccharides from the lignocellulose complex to fermentable sugars represent major costs in the overall process and present a great challenge. Hence, improvements in the development of economically viable technologies for the production of saccharifying enzymes are essential for optimizing the biofuel production process. Topics are summarized from a practical point of view including classification and properties of cellulases, synergistic action, action mechanisms and accessory proteins as well as the future trends for cellulase production, applications and biotechnological perspectives of these enzymes.

A detailed description of the microbial glycoside hydrolases able to degrade lignocellulosic biomass is very important for a better understanding of the new processes involving biorefinery - the conversion of biomass into biofuels. Cellulose and hemicellulose, the major carbohydrates of plant biomass, together with lignin, constitute the most abundant organic compounds present in nature. Cellulose and hemicellulose are converted enzymatically into glucose, xylose or other sugars, which may be fermented by yeasts into second-generation bioethanol or other chemicals. In order to process the lignocellulosic biomass in biorefinery, the use of efficient degrading enzymes is essential for the bioconversion. In this chapter, we describe recent advances in the characterization of glycoside hydrolases, auxiliary activities and synergism between cellulases and accessory proteins involved in cellulose hydrolysis. Furthermore, considering that pretreatments are necessary for efficient biomass degradation and exposition of the lignocellulosic components, a detailed description of several physical, chemical, physicochemical, biological and integrated pretreatments is also presented. Modern and classical methods, including spectroscopy, coupled chromatography, electron microscopy, MALDI- imaging MS, and others, are also discussed as strategies for improving both fiber characterization and understanding of the saccharification of lignocellulose and subsequent biofuel production.

Biofuels derived from lignocellulose are attractive alternative fuels but their production suffers from a costly and inefficient saccharification step that uses fungal enzymes. One route to improve this efficiency is to better understand the transcriptional regulation and responses of filamentous fungi to lignocellulose. Sensing and initial contact of the fungus with lignocellulose is an important aspect. Differences and similarities in the responses of fungi to different lignocellulosic substrates can partly be explained with existing understanding of several key regulators and their mode of action, as will be demonstrated for Trichoderma reesei, Neurospora crassa and Aspergillus spp. The regulation of genes encoding Carbohydrate Active enZymes (CAZymes) is influenced by the presence of carbohydrate monomers and short oligosaccharides, as well as the external stimuli of pH and light. We explore several important aspects of the response to lignocellulose that are not related to genes encoding CAZymes, namely the regulation of transporters, accessory proteins and stress responses. The regulation of gene expression is examined from the perspective of mixed cultures and models that are presented for the nature of the transcriptional basis for any beneficial effects of such mixed cultures. Various applications in biofuel technology are based on manipulating transcriptional regulation and learning from fungal responses to lignocelluloses. Here, we critically access the application of fungal transcriptional responses to industrial saccharification reactions. As part of this chapter, selected regulatory mechanisms are also explored in more detail.

The search for alternatives to conventional fuels becomes more and more important. Second generation biofuels might be a solution to provide us with renewable energy. There are several alternatives to the fossil fuels, such as ethanol, isobutanol, sesquiterpenoids and fatty acid ethyl esters (FAEEs). Saccharomyces cerevisiae is a potent and versatile cell factory, which is able to produce these substances. Except for ethanol the so far achieved yields need to be improved. S. cerevisiae has to be equipped with the proper tools to degrade lignocellulose. This can be achieved by different strategies: secretion or cell surface display of lignocellulose-degrading enzymes, synthesis of cellulosomes or intracellular cellodextrin hydrolysis. Many filamentous fungi possess the ability to efficiently degrade lignocellulose, but they do not produce larger quantities of ethanol due to intolerance towards the product and anaerobic conditions. Therefore, the goal of production of second generation biofuels could also be achieved by enabling them to produce and tolerate higher amounts of ethanol.

Efficient biomass degradation into fermentable sugars is still a major challenge in the biotechnology field. The heterogeneity and structural complexity of the fiber make enzyme action difficult. Fungi belonging to the phylum Ascomycota and Basidiomycota, as pathogenic or saprophytic organisms, are adapted to infect/obtain nutrients from various carbon and nitrogen sources, including several kinds of wood, soil, and organic waste materials. According to genomic, proteomic and biochemical data, the efficiency of fungi to break down plant cell walls is due to their capacity to produce a wide range of CAZymes. These microorganisms have a complex machinery to secrete a broad spectrum of enzymes for releasing carbon and nitrogen locked in complex substrates for nutrition. The majority of proteins secreted by filamentous fungi are glycosylated and their capacity to secrete proteins is probably faster than their synthesis. In spite of their efficiency, the production level of many proteins of interest in natural strains is too low for commercial exploitation. However, industrial strains have shown remarkable improvements in protein secretion yield after traditional mutagenesis techniques. The first part of this chapter is focused on polysaccharide structures and covers the main fungal enzymes used as a strategy for its degradation. Then, a revision on the heterologous expression of hemicellulases by filamentous fungi hosts will be presented, along with the main bottlenecks in fungal heterologous expression. Finally, we provide an extensive revision of the commercial enzymes derived from filamentous fungi systems.

Protein engineering has become the most important tool for enabling industrial application of biocatalysts. Advances in structure-guided methods and novel techniques for directed evolution and high-throughput screening have facilitated the design of enzymes with improved properties such as functional expression, stability, and catalytic activity. The utilization of lignocellulosic biomass for biofuels production is attractive because it is environmentally sustainable. However, commercialization of biomass biofuels requires efficient bioconversion of cellulosic material to sugar, which is largely hampered by biomass recalcitrance. In this chapter, we present the recent application of protein engineering techniques for maximizing the efficiencies of biocatalysts for biofuel production.

The baker’s yeast Saccharomyces cerevisiae is currently the dominant organism for industrial ethanol production due to its inherent general robustness and its long history of successful usage in the fermentation industry. It demonstrates a high rate of fermentation of hexose sugars, very good tolerance to ethanol and to inhibitors in lignocellulosic hydrolysates. On the other hand, baker’s yeast is unable to metabolize pentose sugars, particularly D-xylose, and L-arabinose, which account for more than one third of the total sugars in lignocellulosic biomass. As a result, it cannot be used for efficient lignocellulose based ethanol production. Great progress has been made to develop pentose-fermenting strains of S. cerevisiae through expression of two distinct heterologous pathways. The first pathway relies on expression of the fungal redox pathway that converts D-xylose or L-arabinose to D-xylulose. This approach suffers from the problem of cofactor imbalance, resulting in unnecessary byproduct formation and therefore lower ethanol yield. The second pathway utilizes xylose isomerase that directly isomerizes D-xylose to D-xylulose or a multistep bacterial pathway that converts L-arabinose to D-xylose-5-P. Expression of the latter pathways is proven superior due to higher ethanol yield per consumed sugar. However, the expression of a bacterial pathway especially into industrial yeast strains has been a challenge a.o. due to the lower activities of the heterologous enzymes in yeast. This challenge has been addressed using various strain engineering approaches, including inverse metabolic engineering and evolutionary engineering. No single strain development approach outshines alone. Thus, successful strain development strategies should encompass a combination of the different engineering strategies.

In recent years, the production of renewable fuels such as biodiesel has attracted considerable interest as an alternative to fossil fuels. The conventional alkalicatalyzed processes of biodiesel production present a number of drawbacks to which enzymatic processes present interesting solutions. Fungal lipases are among the most used biocatalysts in the enzymatic synthesis of biodiesel. Different strategies are currently under study in order to reduce the biocatalyst cost and to obtain a more robust biocatalyst with high activity and stability. This article reviews the status and possibilities for biodiesel production using fungal lipases, and discusses the critical aspects that influence lipase activity and stability, including choice of raw material and alcohols, type of biocatalyst, use of solvents, and water activity. Moreover, the development of whole-cells as a new promising technology, the exploration of alternative low-cost oils as potential feedstock and the evaluation of possible reactor configurations are also reported. In conclusion, considerations on the process and an economic efficacy analysis of industrial enzymatic biodiesel production are presented.

In this chapter, we carried out an extensive revision focusing on recent trends on immobilization of microbial enzymes responsible for the degradation of plant cell wall components. We include studies on both individual and mixed enzyme immobilization techniques. Among the diverse techniques, the use of pre-existing supports (chemical or physical binding) and the immobilization without supports enzyme cross-linked aggregates (CLEAs) or crystals (CLECs) were the most promising ones. Nevertheless, the development of new supports with valuable characteristics for the immobilization of these enzymes is still an interesting research field. Furthermore, studies on application of the biocatalysts in a small scale process or in a reactor were also reviewed. In this regard, the lignin component of plant cell walls has been investigated as a support for enzyme immobilization.

Cellulases comprise an important group of enzymes involved in several industrial processes. In recent years they are especially being used in the lignocellulosic biomass conversion into small sugars to produce cellulosic ethanol and chemicals. Filamentous fungi are the most important producers of these catalysts. In order to increase cellulase yield and reduce the production costs, many strategies are being applied such as, submerged fermentation or solid state fermentation using waste residues, aligned with advanced molecular techniques to improve the enzymes performance, reducing its amounts in industrial process as well as the time required by its production. Thus, this chapter will present some of the most important procedures to scale up cellulase production to enable second generation industry.

The most important challenges for conversion of lignocellulosic plant biomass into bioproducts are to overcome its recalcitrance and to reduce steps needed for its biorefining. Conventionally, conversion is carried out using pretreatment followed by the hydrolysis of biomass to monomer sugars that are fermented into different bioproducts. Fungi are able to efficiently degrade biomass by secreting enzymes as well as they are capable of producing a wide range of compounds of commercial interest such as organic acids. The hydrolysis of lignocellulosic biomass requires an efficient cocktail of hydrolytic enzymes, especially cellulases. Commonly, effective cellulases from Trichoderma reesei monoculture are supplemented with separately produced beta-glucosidases from Aspergillus niger. Fungal consortia, resembling microbial combinations synergistically degrading lignocellulosic substrates in nature, may be excellent systems for on-site production of a cocktail of lignocellulolytic enzymes in a single reactor in a biorefinery. Taking this a step further, consolidated bioprocessing has been suggested as an efficient and economical method of manufacturing bioproducts from lignocellulose. The main idea in consolidated bioprocessing is that hydrolysis and fermentation are integrated into a single process, thereby significantly reducing the amount of steps in the biorefinery. A further advance is to also integrate biological pretreatment of biomass in the consolidated process. However, it is not clear whether this would be accomplished more efficiently with microbial consortia or a single organism. Also, biological pretreatment of biomass is still under development. This chapter highlights examples of fungal consortia for production of enzymes and bioproducts, and discusses development of symbiotic mixed cultures for consolidated bioprocessing.

The post-genomic sequencing stimulated all sorts of biological sequencing strategies such as genomic DNA sequencing, mRNA-Seq, LC-MS/MS peptide counting and metabolomics enabling the establishment of complex and interactive networks made out of genes, proteins and metabolites. High-throughput methodologies analyze multiple metabolic pathways simultaneously offering benefits in the exploitation of new biorefining processes. However, it is evident that a single approach cannot unravel or solve the complexities of fundamental microbial biology by just making the integration of multiple information layers. Therefore, efforts have been made to systematically analyze the correlation between transcriptomic and proteomic datasets, improving the chances of capturing gene protein relationships as well as integration of novel biological processes. Here, we describe high-throughput genomics and proteomics tools, discuss the associated challenges and analyze fungal secretomes.

Filamentous fungi are remarkable organisms that are naturally specialized in deconstructing plant biomass. This feature explains their tremendous potential for biofuel production from renewable sources. However, organisms from this group need to be engineered to make them compatible with standard operating procedures of the industry and to optimize their applications in the field of biotechnology. Here, we discuss the development of new tools and approaches for the engineering of fungi for biotech applications such as the production of biofuels. These tools include not only next-generation “omics” tools to obtain deeper insight into the molecular biology of filamentous fungi, but also novel engineering approaches for the genetic modification of these organisms to generate highly efficient cell factories. The first set of tools, in framework of Systems Biology, targets understanding how these fascinating organisms are able to integrate multi-level environmental information in order to coordinate gene expression and protein production in response to changing conditions. The second approach, in the context of Synthetic Biology, provides physical and conceptual tools that allow the genetic modification of fungi, mainly through the construction of synthetic promoters for the expression of heterologous genes in these organisms and either expanding their capabilities or reshaping their regulatory networks. Finally, we discuss some new directions that have been initiated or should be addressed in future work in order to fill gaps still existing in the field.