Trichoderma reesei has been known to be safe when it comes to industrial-scale enzyme production. There are a wide range of applications of xylanases and cellulases that are produced by this fungus. Some of them you can find in animal fee, food, textile, pharmaceutical, and many more. Remember that Trichoderma reesei is non-pathogenic to humans and there is no evidence that it can produce antibiotics or fungal toxins in conditions utilized for enzyme production.
Recently, genetic engineering techniques have been utilized to enhance the industrial production strains of Trichoderma reesei. Besides, with a lot of understanding of safe use of Trichoderma reesei strains, there is an increase in its production. As a result, you can consider its natural enzymes as safe and it’s a safe host for several other gene products.
The traits of filamentous fungi
One of the characteristics of most filamentous fungi is that they can secrete a variety of extracellular proteins and enzymes to interact with their environment. In this way, they use various polymers as energy and carbon sources in their habitats. It’s worth noting that this is a unique property that makes them great hosts for the industrial production of depolymerizing enzymes. There are many fungal species available out there, but only a couple of species are usually utilized for industrial protein production.
The genome of the other fungi species can be exploited to make new enzymes and proteins that may be expressed heterologously in other hosts. However, the product level of some proteins in natural occurring fungi strains tend to be too low for industrial applications. Therefore, strain improvement projects by mutagenesis as well as selection and protein production optimization have been done. As a result, this increased the levels of exported proteins. Today, it is usually expected that Trichoderma reesei industrial strains can produce at least 100g/L.
Filamentous fungi like Trichoderma reesei, Myceiophthora thermophila, and Aspergillus spp. are used as expression platforms for industrial enzyme production. This is because they can produce and secrete large amounts of enzymes. Also, they have a long history of safe use when it comes to industrial enzyme production and can be utilized for large-scale fermentation. You should remember that the production of enzymes is recognized by the FDA.
Fungal protein production
Gene-based studies, such as gene cloning, simple transformation, and many more can be laborious and hard when you decide to use some microbial cell factories like Saccharomyces cerevisiae, Pichia pastoris, and Escherichia coli. Besides this, Neurospora crassa and Aspergillus nidulans have limited applications as biotechnological organisms.
Hence, it’s usually necessary to transfer tools or methods to the fungal factories like Trichoderma reesei. This situation may not change in the short term, but the advent of the various omics technologies, such as transcriptomics and genomics can overcome the challenges in protein production.
There are wild-type genomes as well as genomes of improved producer strains that can use system biological approaches for protein production studies. One of the major benefits of filamentous fungi compared to other microbial factories is that they can produce higher amounts of extracellular enzymes. Therefore, most industrial protein productions with fungi target the outside of the cell. This happens to be a common tactic that aims to enhance translation and posttranslational processes. In such cases, you can genetically fuse the targeted proteins with endogenous protein carriers.
There have been successes for endogenous proteins when you try to use the efficient protein synthesis and export machinery of the industrial fungi. However, the amount of heterologously expressed proteins, particularly non-fungal proteins, can usually be one or two levels lower. Therefore, the tools and methods used to enhance the expression of heterologous proteins in the fungi is not satisfactory.
There are many challenges facing these production processes, though some of them have been managed by modifying the expression host genetically. Some of the solutions to these challenges include introducing several copies of the gene, using strong inducing or constitutive promoters, efficient secretion signals, and constructing protease deficient strains.
These strategies can be ideal when it comes to raising protein yields, but there are still low yields of heterologous protein. This means there are some extra barriers and there is little understanding of the restricting factors. Aside from the protein yield, it’s also crucial to produce the targeted protein in its active form.
You can produce extracellular protein eukaryotes by using a highly specialized secretion pathway that can achieve several functions designed to convert the protein of interest into its active form. This includes folding, a proteolytic process of the mature protein, and adding and processing N- and O-glycans. After the intracellular protein translation, many extracellular proteins can be derived from the endoplasmic reticulum.
There are several things that happen in the endoplasmic reticulum. These include proteolytic processing, the addition of glycans, and folding. This can happen before they get transferred via the Golgi apparatus and exported. You should note that these proteins are further secreted via the hyphal tip.
Recently, it has become evident that certain proteins can follow different routes and can be transferred to other areas of the hyphae, such as septa. Because these proteins go in different ways, there is a chance that it may contribute to the failure or success in recombinant protein production. Another area worth consideration is the role of the molecular foldases and chaperones that help in the non-covalent folding of proteins.
You should note that increased target protein synthesis can lead to overloading of the endoplasmic reticulum. This can cause a down regulation when it comes to expressing the recombinant produced protein as well as other proteins. This can happen by triggering the response of the unfolded proteins or repressing under secretion stress.
Manipulating their expression levels, such as the molecular chaperone BiP can improve the heterologous protein yield by increasing their folding capacity levels. And, the increase in unfolded protein in the endoplasmic reticulum can usually lead to the UPR triggering and can induce the expression of molecular chaperone, vesicular traffic components, and many more. This can resolve the endoplasmic reticulum stress by increasing the transport, folding, and degradation of proteins.