Fungal Cell Factories for Efficient and Sustainable Production of Proteins and Peptides.

Filamentous fungi are a large and diverse taxonomically group of microorganisms found in all habitats worldwide. They grow as a network of cells called hyphae. Since filamentous fungi live in very diverse habitats, they produce different enzymes to degrade material for their living, for example hydrolytic enzymes to degrade various kinds of biomasses. Moreover, they produce defense proteins (antimicrobial peptides) and proteins for attaching surfaces (hydrophobins). Many of them are easy to cultivate in different known setups (submerged fermentation and solid-state fermentation) and their secretion of proteins and enzymes are often much larger than what is seen from yeast and bacteria. Therefore, filamentous fungi are in many industries the preferred production hosts of different proteins and enzymes. Edible fungi have traditionally been used as food, such as mushrooms or in fermented foods. New trends are to use edible fungi to produce myco-protein enriched foods. This review gives an overview of the different kinds of proteins, enzymes, and peptides produced by the most well-known fungi used as cell factories for different purposes and applications. Moreover, we describe some of the challenges that are important to consider when filamentous fungi are optimized as efficient cell factories.

Metabolic engineering of Aspergillus niger via ribonucleoprotein-based CRISPR-Cas9 system for succinic acid production from renewable biomass.

BACKGROUND: Succinic acid has great potential to be a new bio-based building block for deriving a number of value-added chemicals in industry. Bio-based succinic acid production from renewable biomass can provide a feasible approach to partially alleviate the dependence of global manufacturing on petroleum refinery. To improve the economics of biological processes, we attempted to explore possible solutions with a fungal cell platform. In this study, Aspergillus niger, a well-known industrial production organism for bio-based organic acids, was exploited for its potential for succinic acid production. RESULTS: With a ribonucleoprotein (RNP)-based CRISPR-Cas9 system, consecutive genetic manipulations were realized in engineering of the citric acid-producing strain A. niger ATCC 1015. Two genes involved in production of two byproducts, gluconic acid and oxalic acid, were disrupted. In addition, an efficient C4-dicarboxylate transporter and a soluble NADH-dependent fumarate reductase were overexpressed. The resulting strain SAP-3 produced 17 g/L succinic acid while there was no succinic acid detected at a measurable level in the wild-type strain using a synthetic substrate. Furthermore, two cultivation parameters, temperature and pH, were investigated for their effects on succinic acid production. The highest amount of succinic acid was obtained at 35 °C after 3 days, and low culture pH had inhibitory effects on succinic acid production. Two types of renewable biomass were explored as substrates for succinic acid production. After 6 days, the SAP-3 strain was capable of producing 23 g/L and 9 g/L succinic acid from sugar beet molasses and wheat straw hydrolysate, respectively. CONCLUSIONS: In this study, we have successfully applied the RNP-based CRISPR-Cas9 system in genetic engineering of A. niger and significantly improved the succinic acid production in the engineered strain. The studies on cultivation parameters revealed the impacts of pH and temperature on succinic acid production and the future challenges in strain development. The feasibility of using renewable biomass for succinic acid production by A. niger has been demonstrated with molasses and wheat straw hydrolysate.

Metabolic specialization in itaconic acid production: a tale of two fungi.

Some of the oldest and most established industrial biotechnology processes involve the fungal production of organic acids. In these fungi, the transport of metabolites between cellular compartments, and their secretion, is a major factor. In this review we exemplify the importance of both mitochondrial and plasma membrane transporters in the case of itaconic acid production in two very different fungal systems, Aspergillus and Ustilago. Homologous and heterologous overexpression of both types of transporters, and biochemical analysis of mitochondrial transporter function, show that these two fungi produce the same compound through very different pathways. The way these fungi respond to itaconate stress, especially at low pH, also differs, although this is still an open field which clearly needs additional research.

Application of lactic acid bacteria in green biorefineries.

Lactic acid bacteria (LAB) have extensive industrial applications as producers of lactic acid, as probiotics, as biocontrol agents and as biopreservatives. LAB play a large role in food fermentation and in silage processes, where crops such as grass, legumes, cereals or corn are fermented into high-moisture feed that is storable and can be used to feed cattle, sheep or other ruminants. LAB also have great applications within green biorefineries, with simultaneous production of protein-rich feed for monogastric animals, silage or feed pellets for ruminants and production of lactic acid or specific amino acids. In green biorefineries, fresh or ensiled wet biomass is mechanically fractionated into green juice and solid residues (press cake), where the plant juice, for example, can be used for production of lactic acid using LAB. In a process named 'ENLAC', recovery of protein and chlorophyll from silage by simultaneous lactic acid fermentation and enzyme hydrolysis has been developed. Furthermore, a process for protein recovery was recently developed by applying a specific LAB starter culture to green juice from freshly harvested crops. This paper focuses on reviewing LAB for their applications within biorefining of 'green' crops such as clover, alfalfa, grasses and other green plant materials.

Discovery of a Novel Fungus with an Extraordinary ß-Glucosidase and Potential for On-Site Production of High Value Products.

Among cellulases, ß-glucosidases play a key role in the final conversion of cellulose into glucose as well as they boost the performance of the other cellulases, in particular cellobiohydrolases in relieving product inhibition. This chapter serves as case example from screening for novel fungal cellulases focusing on ß-glucosidases to identifying a gene encoding the key ß-glucosidase in the fungus with highest activity. In the case example, the ß-glucosidase-producing fungus showed to belong to an unknown fungal species, Aspergillus saccharolyticus, not previously described. The gene was expressed in Trichoderma reesei, which has low indigenous ß-glucosidase activity, and the activity of the purified enzyme was assessed in hydrolysis of various pretreated lignocellulosic biomasses. The potential of using the natural producing strain for on-site production of ß-glucosidases using lignocellulosic biorefinery waste streams as substrates is discussed. Finally, the potential of the fungus for consolidated bioprocessing of waste streams into valuable compounds, such as organic acids is highlighted.

Isolation and Screening of Cellulolytic Filamentous Fungi.

Filamentous fungi are among the microorganisms that most efficiently are able to degrade plant biomass by secreting cell wall-degrading enzymes and they are therefore used extensively in the industry as workhorses for the production of enzymes, including cellulases for the use in second-generation biorefinery concepts. Fungi are therefore of interest both as resources for the search of novel cellulolytic enzymes and for production of enzymes and enzyme cocktails, which also can be carried out on-site using cheap lignocellulosic substrates for growth and enzyme production. Fungi can be isolated from different environmental niches, such as soil, compost, decaying wood, decaying plant material, building materials, and different foodstuffs. Selective isolation can be carried out using simple cellulosic or complex plant material in the media. In this chapter, methods used for the isolation and screening of cellulolytic fungi isolated from different ecological niches are presented. The screening assay presented in the chapter is an easy semiquantitative high-throughput agar plate screening method using azurine-cross-linked (AZCL) cellulose substrates.

Overexpression of a C4-dicarboxylate transporter is the key for rerouting citric acid to C4-dicarboxylic acid production in Aspergillus carbonarius.

BACKGROUND: C4-dicarboxylic acids, including malic acid, fumaric acid and succinic acid, are valuable organic acids that can be produced and secreted by a number of microorganisms. Previous studies on organic acid production by Aspergillus carbonarius, which is capable of producing high amounts of citric acid from varieties carbon sources, have revealed its potential as a fungal cell factory. Earlier attempts to reroute citric acid production into C4-dicarboxylic acids have been with limited success. RESULTS: In this study, a glucose oxidase deficient strain of A. carbonarius was used as the parental strain to overexpress a native C4-dicarboxylate transporter and the gene frd encoding fumarate reductase from Trypanosoma brucei individually and in combination. Impacts of the introduced genetic modifications on organic acid production were investigated in a defined medium and in a hydrolysate of wheat straw containing high concentrations of glucose and xylose. In the defined medium, overexpression of the C4-dicarboxylate transporter alone and in combination with the frd gene significantly increased the production of C4-dicarboxylic acids and reduced the accumulation of citric acid, whereas expression of the frd gene alone did not result in any significant change of organic acid production profile. In the wheat straw hydrolysate after 9 days of cultivation, similar results were obtained as in the defined medium. High amounts of malic acid and succinic acid were produced by the same strains. CONCLUSIONS: This study demonstrates that the key to change the citric acid production into production of C4-dicarboxylic acids in A. carbonarius is the C4-dicarboxylate transporter. Furthermore it shows that the C4-dicarboxylic acid production by A. carbonarius can be further increased via metabolic engineering and also shows the potential of A. carbonarius to utilize lignocellulosic biomass as substrates for C4-dicarboxylic acid production.

The global regulator LaeA controls production of citric acid and endoglucanases in Aspergillus carbonarius.

The global regulatory protein LaeA is known for regulating the production of many kinds of secondary metabolites in Aspergillus species, as well as sexual and asexual reproduction, and morphology. In Aspergillus carbonarius, it has been shown that LaeA regulates production of ochratoxin. We have investigated the regulatory effect of LaeA on production of citric acid and cellulolytic enzymes in A. carbonarius. Two types of A. carbonarius strains, having laeA knocked out or overexpressed, were constructed and tested in fermentation. The knockout of laeA significantly decreased the production of citric acid and endoglucanases, but did not reduce the production of beta-glucosidases or xylanases. The citric acid accumulation was reduced with 74-96 % compared to the wild type. The endoglucanase activity was reduced with 51-78 %. Overexpression of LaeA seemed not to have an effect on citric acid production or on cellulose or xylanase activity.

Co-cultivation of Trichoderma reesei RutC30 with three black Aspergillus strains facilitates efficient hydrolysis of pretreated wheat straw and shows promises for on-site enzyme production.

Co-cultivation of fungi may be an excellent system for on-site production of cellulolytic enzymes in a single bioreactor. Enzyme supernatants from mixed cultures of Trichoderma reesei RutC30, with either the novel Aspergillus saccharolyticus AP, Aspergillus carbonarius ITEM 5010 or Aspergillus niger CBS 554.65 cultivated in solid-state fermentation were tested for avicelase, FPase, endoglucanase and beta-glucosidase activity as well as in hydrolysis of pretreated wheat straw. Around 30% more avicelase activity was produced in co-cultivation of T. reesei and A. saccharolyticus than in T. reesei monoculture, suggesting synergistic interaction between those fungi. Fermentation broths of mixed cultures of T. reesei with different Aspergillus strains resulted in approx. 80% efficiency of hydrolysis which was comparable to results obtained using blended supernatants from parallel monocultures. This indicates that co-cultivation of T. reesei with A. saccharolyticus or A. carbonarius could be a competitive alternative for monoculture enzyme production and a cheaper alternative to commercial enzymes.

Advancing USER cloning into simpleUSER and nicking cloning.

The two novel methods for DNA cloning presented here have been developed for the rapid construction of vectors used for insertion of genes in filamentous fungi. The current study shows that both simpleUSER cloning and nicking cloning can substitute USER cloning for insertion of single PCR fragments into plasmids. The simpleUSER cloning method proposed in this paper varies from USER cloning by substituting the dual enzymatic plasmid preparation step with a single enzymatic step. The other method further abolishes the use of USER™ enzyme mix and PfuTurbo Cx polymerase, and is referred to as nicking cloning. We show that both simpleUSER cloning and nicking cloning can substitute USER cloning for insertion of single PCR fragments into plasmids, and that the combination of these two methods works efficiently for the construction of selective plasmids and plasmids for co-transformation. This strategy was applied to genetically modify the filamentous fungus Aspergillus carbonarius. The two methods simplify DNA cloning by reducing time and complexity associated with cloning in filamentous fungi.

ß-glucosidases from a new Aspergillus species can substitute commercial ß-glucosidases for saccharification of lignocellulosic biomass.

ß-Glucosidase activity plays an essential role for efficient and complete hydrolysis of lignocellulosic biomass. Direct use of fungal fermentation broths can be cost saving relative to using commercial enzymes for production of biofuels and bioproducts. Through a fungal screening program for ß-glucosidase activity, strain AP (CBS 127449, Aspergillus saccharolyticus ) showed 10 times greater ß-glucosidase activity than the average of all other fungi screened, with Aspergillus niger showing second greatest activity. The potential of a fermentation broth of strain AP was compared with the commercial ß-glucosidase-containing enzyme preparations Novozym 188 and Cellic CTec. The fermentation broth was found to be a valid substitute for Novozym 188 in cellobiose hydrolysis. The Michaelis-Menten kinetics affinity constant as well as performance in cellobiose hydrolysis with regard to product inhibition were found to be the same for Novozym 188 and the broth of strain AP. Compared with Novozym 188, the fermentation broth had higher specific activity (11.3 U/mg total protein compared with 7.5 U/mg total protein) and also increased thermostability, identified by the thermal activity number of 66.8 vs. 63.4 °C for Novozym 188. The significant thermostability of strain AP ß-glucosidases was further confirmed when compared with Cellic CTec. The ß-glucosidases of strain AP were able to degrade cellodextrins with an exo-acting approach and could hydrolyse pretreated bagasse to monomeric sugars when combined with Celluclast 1.5L. The fungus therefore showed great potential as an onsite producer for ß-glucosidase activity.