REVIEW         

Biotechnological tools for genetic improvement of Trichoderma

Liliana Villao-Uzho1 ; Fernando Espinoza-Lozano1 ; Luis Galarza-Romero1, 2 ;

Efrén Santos-Ordóñez1, 2, *

1  Centro de Investigaciones Biotecnológicas del Ecuador, ESPOL Polytechnic University, ESPOL, Campus Gustavo Galindo, km 30.5 Vía Perimetral, 090902, Guayaquil, Ecuador.

2  Facultad de Ciencias de la Vida, ESPOL Polytechnic University, ESPOL, Campus Gustavo Galindo, km 30.5 Vía Perimetral, 090902, Guayaquil, Ecuador.

* Corresponding author: gsantos@espol.edu.ec (E. Santos-Ordóñez).

Received: 25 September 2023. Accepted: 23 March 2024. Published: 8 April 2024.

Abstract

Trichoderma is a cosmopolitan fungus widely distributed around the world. The different uses of this beneficial fungus are varied in several industries, like agriculture, textile, and paper, including the pharmaceutical industry. The genus Trichoderma has other mechanisms of action, including the production of different enzymes and secreted secondary metabolites used in various industries. The genomes of different Trichoderma species have been sequenced to identify the mechanisms for producing several compounds. The advancement of multiple technologies has allowed the development of transformation tools for the genetic improvement of Trichoderma, thus increasing biomass, primary and secondary metabolites, and enzymes. Therefore, genetic modification aims to increase compound production in several Trichoderma strains. Characterization of Trichoderma through gene expression analysis is essential for biotechnology applications. It helps counteract one of the most challenging problems for agriculture today, including climate change and the appearance of pathogens that attack crops with high commercial and food demand. In conclusion, this review analyzes various strategies to improve Trichoderma strains genetically and their multiple applications in the agricultural, textile, paper, and pharmaceutical industries. As a recommendation for future studies with potential impact, the optimization of specific genetic modifications in Trichoderma strains is recommended to improve their adaptability and effectiveness in combating emerging challenges in agriculture, especially those linked to climate change. Investigating possible synergies between genetically modified Trichoderma strains and environmentally sustainable agricultural practices could contribute to developing solutions for crop protection and yield improvement.

Keywords: metabolites; genetic transformation; molecular tools; Trichoderma.

DOI: https://dx.doi.org/10.17268/sci.agropecu.2024.016

Cite this article:

Villao-Uzho, L., Espinoza-Lozano, F., Galarza-Romero, L., & Santos-Ordóñez, E. (2024). Biotechnological tools for genetic improvement of Trichoderma. Scientia Agropecuaria, 15(2), 213-223.

1. Introduction

The rise in population over the decades has neces­sitated meeting a significant food demand, leading to the expansion of substantial areas dedicated to commercial crops. Consequently, enhancing agri­cultural yield, growth, or production has become imperative, prompting the utilization of broad-spectrum chemical fertilizers, which have even approached the permissible human limit. (Gladek et al., 2017).

In sustainable agricultural practices, the manage­ment of plant diseases is crucial; various fungi from different genera have proven to be efficient biocon­trol agents against phytopathogenic fungi, with Trichoderma standing out for its extensive use in agriculture (Guzmán-Guzmán et al., 2023). Incor­porating these biocontrol agents into farming practices contributes to environmentally friendly disease management strategies, facilitating decreased chemical usage and promoting a more sustainable approach to preserving crop health (El-Saadony et al., 2022; Yao et al., 2023).

Meanwhile, in the pursuit of solutions to address the impending population growth and the pressing need for field practices aligned with food safety regulations, pertinent information has emerged re­garding the efficacy of biocontrollers, in the case of utilizing microencapsulated derived from Tricho­derma isolates, which has proven to be a valuable strategy in managing diseases, notably effective against Moniliopthora roreri. This pathogen has posed a significant threat to cocoa crops, particularly in countries like Ecuador and Colombia, where it has caused substantial damage (Valenzuela-Cobos et al., 2023). Applying these beneficial fungi through microencapsulation has shown promising results (Qi et al., 2023), significantly mitigating the development of the disease and providing a practical solution to combat the destructive impact of Moniliopthora roreri on cocoa cultivation.

This innovative approach not only demonstrates the efficacy of Trichoderma isolates but also under­scores the potential of microencapsulation as a tar­geted and efficient delivery method for enhancing the biocontrol capabilities of beneficial fungi in ag­riculture (Avilés et al., 2023; Maruyama et al., 2020).

Phytochemical studies have delved into com­pounds that function as biocontrol agents. Recent studies reveal that the inhibitory activity may be as­sociated with various chemical components, includ­ing alkaloids, lactones, quinones, flavonoids, and antifungal compounds. Some examples identified through gas chromatography-mass spectrometry include widdrol, β-caryophyllene, tyrosol, isobutyrate, and butyl sorbic acid. This extensive examination sheds light on the diverse chemical mechanisms underlying the inhibitory effects, offer­ing valuable insights into the potential applications of these bioactive compounds in agricultural dis­ease management (Chóez-Guaranda et al., 2023).

In addition to acting as defenders against diverse pathogens, fungal biocontrol agents safeguard plants from various forms of abiotic stress, support­ing plant growth and production (Fazeli-Nasab et al., 2022). An additional aspect regarding Trichoderma is the potential utilization of its various secondary metabolites. These metabolites have chemical characteristics that have attracted signifi­cant attention in the industrial, pharmaceutical, and food sectors (Zhang et al., 2021). Consequently, numerous studies are currently being carried out to explore the various metabolites and discover their excellent efficacy against phytopathogens (Guo et al., 2023; Khan et al., 2020). This exploration not only underlines the versatility of Trichoderma but also hints at its promising applications in various industries, showing its potential as a valuable resource for developing novel products with agricultural, medical, or food-related benefits.

This review presents an overview of the present status and historical development of Trichoderma spp., highlighting the importance of employing bioinputs as a biotechnological tool for enhancing agriculture. It underscores the validation and promotion of genetic improvement in various strains of filamentous fungi through molecular mechanisms to achieve advancements in plant growth and to improve their resilience against phytopathogenic agents.

2. Overview of Trichoderma spp

The species of the genus Trichoderma have rapid growth, showing green conidia with a branched structure (Schmoll et al., 2010) (Figure 1). They are present in agricultural soils and forests, encompassing diverse lifestyles and interactions with other organisms or adapting to inhospitable and sterile soils such as bioreactors or fermenters, which makes them attractive in the current climate change environment (Yao et al., 2023).

Figure 1. Trichoderma spp., morphology; A) and B) phialides, C) spores D) Conidiophores.

Along with their different roles in various habitats, Trichoderma strains have been described as biological controllers of diseases caused by pathogens in different crops due to their other mechanisms of action (Figure 2) (Galarza et al., 2015; Yao et al., 2023). These mechanisms recognize the attack by pathogens through the rupture of the cell wall and the absorption of the released nutrients, denominated mycoparasitism (Tyśkiewicz et al., 2022). This interaction with pathogens induces the local or systemic resistance of the plant (elicitors, endoelicitors, exoelicitors) and mainly in the secretion of enzymes and secondary metabolites; each strain may have its mechanism of interaction between the pathogen and the host plant (Tyśkiewicz et al., 2022; Zin et al., 2020).

The Trichoderma genus involved several species with bioprospecting and biotechnology applications focused on the high capacity of enzyme production, with different applications, especially in agriculture to control plant pathogen disease, in biofuel production, degradation of clothing in the textile industry (Abd et al., 2018; Carvalho et al., 2017). The significance and potential environmental impact of specific genes have prompted a shift towards sustainable alternatives in agriculture. Given the widespread use of synthetic products like herbicides and fertilizers, which may pose environmental and health risks, there is a growing interest in exploring natural solutions (Çakmakçı et al., 2023). Trichoderma, known for its remarkable adaptive capacity to diverse environmental conditions, has emerged as a promising source of metabolites and bio-controllers (Sandle, 2014). These alternatives offer effective pest and pathogen control and align with the principles of environmentally friendly and sustainable agriculture (Abdul-Halim et al., 2023).

In practical terms, research has demonstrated the inhibitory activity of Trichoderma species, particularly Trichoderma asperellum and Trichoderma atroviride, against various pathogens such as Fusarium and Aspergillus. These pathogens have been responsible for significant agricultural losses (Guzmán-Guzmán et al., 2023). Using Trichoderma-based solutions presents a valuable opportunity to mitigate the adverse effects of harmful synthetic chemicals, contributing to the development of more eco-friendly and resilient agricultural practices. (Stracquadanio et al., 2020).

In other aspects, some varieties of Trichoderma have been used to combat diseases in cocoa (Theobroma cacao), such as Vascular streak dieback (VSD) caused by Oncobasidium theobromae due to the high potentiality of the secondary metabolites in combination with organic fertilizers (Rosmana et al., 2019). Excellent results have been achieved in terms of the growth of the plants and the number of shoots (Simamora et al., 2021).

In addition, the perspectives of Trichoderma use in different areas lead to improving or optimizing the application of the other species of this genus. Genome analysis of various species revealed a pool of chitinolytic enzymes produced in different stages of the fungus life cycle (Chung et al., 2022). The number of biosynthetic pathways Trichoderma uses to generate other compounds is due to the complexity of its genome (Schalamun et al., 2022). The molecular processes of sequencing and annotation of the Trichoderma reesei genome began further research on this beneficial fungus (Martinez et al., 2008); more than 25 Trichoderma genomes have been added (Kubicek et al.,2019; Mukherjee et al., 2013). Likewise, the genomic studies of this genus were based on genes related to the production of enzymes to obtain biofuel, while T. reesei has been one of the most studied species, which was transformed by selection methods, getting the strain RUT-C30 which produced a high amount of extracellular cellulose (De Paula et al., 2018).

Figure 2. Main biological control mechanisms of Trichoderma spp.

Consequently, and due to its significant capacity to produce enzymes, this species has received Generally Recognized as Safe (GRAS) status granted by the US Food and Drug Administration US Department of Health and Human Services (Liu et al., 2023; Paloheimo et al., 2016). On the other hand, several methodologies have been imple­mented to understand the different mechanisms used by this genus. Different Trichoderma strains have been transformed over time using the polyethylene glycol (PEG), CaCl2, method biolistic (Lorito et al., 1993), electroporation (Goldman et al., 1990), shock waves (Magaña-Ortíz et al., 2013), as well as transformation by Agrobacterium tumefaciens (Zeilinger, 2004) (Figure 3).

T. reesei is a filamentous fungus, and it is among the Trichoderma strains with the most significant utility in the industrial field due to the production of cellulases, hemicellulases, and recombinant proteins (Wu et al., 2019) serving as a host organism to produce recombinant enzymes is one of its prominent applications (Arai et al., 2023). Much of these are used to depolymerize biomass into simple sugars, converted into intermediate chemicals and biofuels, such as ethanol (Martinez et al., 2008; Xu et al., 2015). Similarly, T. reesei has been employed in the biodelignification and subsequent enzymatic hydrolysis of Elephant grass, thereby enabling bioethanol production (Iyyappan et al., 2023).

Recently, with the development of omics tools, it was possible to advance with different studies related to the physiology and biology of the fungus, which is integrated into a biotechnological system looking for complex interactions in the production of enzymes by the strains of the Trichoderma genus, through molecular transformations tools (Cai et al., 2021; Wang et al., 2022). Additionally, genomic, transcriptomic, and proteomic studies allow the characterization and identification of Trichoderma strains related to different applications, increasing our knowledge about the biology of this genus (Wilson et al., 2009).

Figure 3. Different types of genetic transformation of Trichoderma spp.

3. Genomic research

Twenty-five genomic sequences are available from species of the genus Trichoderma, which have opened a range of possibilities to deepen the ben­efits of the species belonging further genetically to this genus. With the aid of this information, func­tional analysis of predicted genes involved in the biological processes of the beneficial fungus is needed. The functional characterization of the genes involved in these processes could be per­formed through knockouts of the different genes. In addition to studying proteins, identification focuses on the interaction routes, including genetic overexpression. Additionally, gene substitution us­ing homologous recombination is considered one of the most used methodologies for characterizing genes of the species under study (Kück et al., 2010).

Analyzes dedicated to the function of the genes of the beneficial Trichoderma species are generally attributed to the construction of recombinant DNA using conventional cloning techniques, which are based on digestion and ligation procedures. This general vector technique for creating deletions or overexpressing mutant strains of genes is consid­ered a technique or methodology that generates disadvantages (Fitz et al., 2018). In contrast, the metabolite's production of enzymes by the Trichoderma genus is one of the primary factors in using this beneficial fungus in several industries.

In this sense, knowing the pathways involved in making this kind of compound is essential. Addi­tionally, in general, two genes could be involved in metabolic pathways; one gene plays a role in the inactivation of the path, while the other gene could be involved in the production of target molecules (Saravanakumar et al., 2020), which could generate the independent activation of a gene that partici­pates precisely in the production of metabolites (Steen et al., 2010).

Likewise, within the production of microbial metab­olites, the coenzymes NADH, ATP, and NADPH are related, which could trigger the production of pri­mary metabolites. Therefore, the genetic modifica­tion of the genes associated with the expression of coenzymes would increase the production of me­tabolites (Zhang et al., 2007). Finally, to avoid inhib­iting the growth of a microorganism, the produc­tion of metabolites must be generated in time (Yazawa et al., 2013). The genomic organization, to­gether with the regulation and enzymatic hydrolytic expression in the Trichoderma harzianum strain IOC3844, are closely related, which helps to in­crease plant degradation (Ferreira Filho et al., 2020).

On the other hand, the Trichoderma genus produces many volatile compounds and enzymes, such as glucanases, chitinases, and proteases (Stoppacher et al., 2010) (Figure 4). The production of enzymes is related to the expression of a battery of genes. Among the species whose genes are associated with the production of enzymes have been characterized and identified, T. harzianum is related to the production of GlcNAcases, while T. atroviride and T. virens are related to the functional genes exc1, exc2, tvnag1, and tvnag2, chi33, chi37, and chi42 (Harman et al., 2004; Howell, 2003).

Figure 4. Chitinolytic activity of several Trichoderma strains.

The genes bgn131, lam1.3, and glu78 are also responsible for glucanase production in the T. harzianum strain (Donzelli et al., 2001). In addition, the genes Prb1 and tvsp1 are related to protease production in the species above. The same author mentioned that T. atroviride and T. virens produce glucanases involved with the genes Tv-bgn1 and Tv-bgn2. Similarly, it was reported that the gene responsible for synthesizing trypsin-like acidic serine peptidase in the T. harzianum strain is pra1 (Suarez et al., 2004). Regarding the mycoparasitism of T. harzianum (Szabó et al., 2013) express that there are genes that are co-expressed during these mechanisms of action, including the sedolisin serine protease p5216, the acidic serine protease pra1, aspartic proteases p6281 and the metalloendopeptidase p7455.

Regarding the secretion of secondary metabolites by the different species of this beneficial fungus, the tex1 gene is mentioned, which is involved in synthesizing peptaiboltrichovirin II (Wiest et al., 2002). In addition, in the T. virens strain, several genes were identified that are related to the production of peptaibol synthetase, including tex1, tex2, and tex3. Furthermore, SirP and GliP clusters are associated with the synthesis of epidithio-dioxopiperazines. The tri5 gene is related to the synthesis of trichothecene; likewise, this strain may have a gene similar to SirP, which is associated with the production of secondary metabolites that have not yet been identified (Howell et al., 2008; Mukherjee et al., 2012).

On the other hand, the T. reesei species faces the presence of the GliP cluster, even though this species does not produce gliotoxin. Several PKS genes have been described in different strains; in the T. harzianum case, ksT-1 and pksT-2 have been reported (Yao et al., 2016). On the other hand, the deletion in a T. reesei strain in the pks4 gene affected processes such as conidia pigmentation, the production of secondary metabolites, and the stability of the cell wall, as well as the antagonistic capacity of the beneficial strain (Atanasova et al., 2013).

4. Genetic Improvement of Trichoderma

Using molecular techniques to develop different stable and reproducible modifications in various mi­croorganism's genomes has benefited both the agricultural and industrial sectors. The Trichoderma genus is known for the highest production of cellulolytic enzymes, such as proteases, cellulases, hemicelluloses, xylanases, amylases, and cellobio­hydrolase, in addition to secondary metabolites with pharmacological properties, such as gliotoxin and gliovirin, peptaibols, polyketides, siderophores, pyrons, and terpenoids/steroids (Mukherjee, Buensanteai, et al. 2012; Zeilinger et al., 2016).

One of the most common techniques is genetic transformation through protoplasts mediated by Agrobacterium tumefaciens (Wang et al., 2019); however, initially for transforming Trichoderma, techniques including electroporation or biolistic were used (Schuster et al., 2016). T. reesei has excellent protein production capacity compared to other microbial systems such as Saccharomyces cerevisiae and Escherichia coli. Due to this, there is great interest in improving these strains for industrial applications in increasing biomass and biofuel production (Martinez et al., 2008).

Figure 5. Trichoderma harzianum GFP morphology red arrows phialides blue arrow conidiophore.

Table 1

CRISPR technology applied to Trichoderma

Furthermore, although the T. reesei genome has been sequenced (Li et al., 2017), little-studied cellular functions are essential in different gene biological pathways. However, despite the multiple utilities of this fungus, some factors limit the production of these enzymes. One of them is the pH since it must be acidic for high enzymatic activity; otherwise, it will diminish Trichoderma activity (Wang et al., 2014). These reasons have led to the development several molecular techniques to increase or maintain the production of enzymes and proteins in T. reesei and other Trichoderma species under different test conditions.

The primary industries for using Trichoderma come from the agricultural sector, so the mechanisms of action focused on knowing and studying various beneficial strains on pathogenic strains and the genetic transformation of indicator genes, including genes that code for green fluorescents protein (GFP) and β-glucuronidase (GUS), which are widely used to analyze the activity of different beneficial genes, especially in agricultural settings (Bae et al.,2000; Green et al., 1995) (Figure 5).

Regarding the improvements of the strains of the beneficial fungus, there are different methodolo­gies widely used to date, including Agrobacterium-mediated transformation, protoplasts, polymerase chain reaction (PCR), SceI meganuclease, zinc finger nuclease, RNA silencing (RNAi), transcription activator-like effector nuclease (TALENs) and more recently the use of CRISPR-Cas9 in filamentous fungi (Shi et al., 2017) (Table 1).

In the transformation mediated by Agrobacterium tumefaciens, only a fraction of the plasmid DNA is randomly integrated into the fungal genome, conferring a transformation efficiency of 100 to 1000 times more than other conventional methods (Cardoza et al., 2006). Another alternative is using vectors or recombinant plasmids to find strategies to explore various gene functions. This process has disadvantages: cloning and ligating the plasmid or inserting it in different genome sites. Recombination efficiency in filamentous fungi is generally low at 5%, with a low percentage of genetic markers (Kück et al., 2010). Various investigations have been carried out to overcome these limitations, such as cloning processes, including Gateway and Golden Gate (Nogueira-López et al., 2019), or as previously mentioned, transformation with protoplasts also results in a widely used technique, where its efficiency ranges between 1 and 103 transformants/µg of DNA (Li et al., 2017).

In turn, this situation has meant a significant advance in our attempt to modify fungal strains genetically, allowing scientists to seek new techniques that enable greater specificity between the different existing fungal strains (Martín, 2015). The rapid progress and broad application of a versatile genome editing technology, namely the CRISPR (regularly interspaced clustered short palindromic repeat)-Cas9 (CRISPR-related nuclease 9) system, revolutionized biological research and has many innovative applications in a wide range of fields that show great promise in the study and application of filamentous fungi such as Trichoderma (Wang et al., 2022).

The CRISPR-Cas9 tool was first used in the T. reesei strain through gRNA by in vitro transformation (Li et al., 2017) (Liu et al., 2015), and gene disruption was achieved through direct in vitro transformation by assembling a complete Cas9/gRNA in the T. reesei strain (Hao and Su, 2019; Rantasalo et al., 2019). However, expression during in vitro gRNA synthesis is not convenient, and the stability of gRNA during transformation could influence the efficiency of genomic editing (Pohl et al., 2016). On the other hand, the gRNA system in different species of filamentous fungi is driven by the RNA polymerase III motor, such as the U6 promoter. Additionally, the U6 snRNA promoter has been widely used in gRNA transcription during transcription using the CRISPR-Cas9 system (Arazoe et al., 2015; Katayama et al., 2016; Zhang et al., 2016; Zhang et al., 2016). Wu et al. (2020) performed in vivo transcription using gRNA synthesized under the control of T. reesei snRNA promoters, causing a ura5 frameshift mutation through non-homologous recombination events presenting a simple editing method of genes using an in vivo gRNA transcription system coupled to CRISPR-CAS9 (Wu et al., 2020).

In the view to reduce undesired enzyme production from T. reesei, three significant cellulitic genes, cellobiohydrolase II (cbh2), endonuclease I and II (egl1 and egl2, respectively), were deleted and replaced with lipase B from Candida Antarctic CalB gene (Rantasalo et al., 2019). On the other hand, T. harzianum is recognized for its various agricultural applications, produces 6-pentyl-pyrone on the transformed strain through the THCTF1 factor, different from the ThMBF1 transcription coactivator which was overexpressed into T. harzianum, causing decrease on the antifungal activity against Fusarium oxysporum and Botrytis cinereal (Rubio et al., 2017).

The study of different genes of Trichoderma strains and their applications are patented for use as biofungicides and in different breeding strategies. Gene editing technologies are essential for genet­ically characterizing this species, although they are still being researched. What has been confirmed are several mutants obtained employing an additional marker in this species using the CRISPR system, biolistically verifying that the alteration of the pyr4 gene does not interfere with the activity of biological control of pathogens (Vieira et al., 2021).

5. Current and future challenges

Trichoderma has many strains that can produce a battery of high-value compounds, such as second­ary metabolites and enzymes. This beneficial fungus appears as a cellular bio-factory, acting as homolo­gous or heterologous, creating various combina­tions of industrial and medical importance. To have better knowledge and understanding of the biolog­ical and metabolic functions of the genus Trichoderma, through genetic engineering tools, researchers can generate genetic modifications related to the production of primary and secondary metabolites, as well as enzymes, which are involved in the homology and heterology of the genetic material of the fungus. In this sense, this chapter provides essential information on genetic manipu­lation tools and functional metabolomics based on different species of the genus Trichoderma related to the high production of various compounds of in­dustrial interest. Despite significant advances, chal­lenges remain, such as the pH sensitivity of Trichoderma, which limits enzyme activity. Ongoing research focuses on developing molecular tech­niques to overcome these limitations and maintain or increase enzyme and protein production under different conditions.

Conclusions

This article reviews the significance of species like Trichoderma, renowned for its rapid growth and di­verse interactions in various environments, encompassing agricultural soils and specialized conditions like bioreactors or fermenters. Trichoderma employs mechanisms such as mycoparasitism, plant resistance induction, and the secretion of enzymes and secondary metabolites to combat phytopathogens.

One of its notable features is its potential in bioprospection and biotechnological applications, particularly in enzyme production for controlling pathogenic plant diseases, biofuel production, and textile industry processes. Various genetic modifica­tion techniques, including Agrobacterium-mediated transformation, protoplasts, and advanced CRISPR-Cas9 technology, enhance strains for industrial applications.

The revolutionary CRISPR-Cas9 system has trans­formed biological research, allowing precise ge­nome editing in filamentous fungi like Trichoderma. This system has been successfully employed to de­lete and replace specific genes, thereby improving the understanding and manipulation of Trichoderma strains for diverse purposes. T. reesei as an example; its substantial utility in the industrial field stems from its production of cellulases, hemicellulases, and recombinant proteins. Each of these benefits has significantly been amplified with the gene editing system. Trichoderma is an environmentally friendly alternative, contributing to more ecological and resilient agricultural practices.

In conclusion, the text highlights the multifaceted nature of Trichoderma, emphasizing the im­portance of genetic exploration, functional analysis, and advanced molecular techniques in unlocking its potential for various applications. Ongoing ad­vancements in genetic modification technologies, particularly CRISPR-Cas9, offer promising prospects for further enhancing and tailoring Trichoderma strains to specific needs, from agriculture to indus­trial processes.

ORCID

L. Villao-Uzho  https://orcid.org/0000-0002-2677-1415

F. Espinoza-Lozano  https://orcid.org/0000-0002-2051-2682

L. Galarza-Romero  https://orcid.org/0000-0002-2870-4080

E. Santos-Ordóñez  https://orcid.org/0000-0002-0749-6024

References

Abd, A. A., Saleh, S. A. A., Eid, B. M., Ibrahim, N. A., & Mostafa, F. A. (2018). Thermodynamics characterization and potential textile applications of Trichoderma longibrachiatum KT693225 xylanase. Biocatal Agric Biotechnol, 14, 129-137. https://doi.org/10.1016/J.BCAB.2018.02.011

Abdul-Halim, A. M. A. A., Shivanand, P., Krishnamoorthy, S., & Taha, H. (2023). A review on the biological properties of Trichoderma spp. as a prospective biocontrol agent and biofertilizer. J Appl Biol Biotechnol, 11(5), 34-46 https://doi.org/10.7324/JABB.2023.11504

Arai, T., Wada, M., Nishiguchi, H., Takimura, Y., & Ishii, J. (2023). Inducer-free recombinant protein production in Trichoderma reesei: secretory production of endogenous enzymes and heterologous nanobodies using glucose as the sole carbon source. Microb Cell Fact, 22, 103. https://doi.org/10.1186/s12934-023-02109-y

Arazoe, T., Miyoshi, K., Yamato, T., Ogawa, T., Ohsato, S., Arie, T., & Kuwata, S. (2015). Tailor-made CRISPR/Cas system for highly efficient targeted gene replacement in the rice blast fungus. Biotechnol Bioeng, 112(12), 2543-2549. https://doi.org/10.1002/bit.25662

Atanasova, L., Knox, B. P., Kubicek, C. P., Druzhinina, I. S., & Baker, S. E. (2013). The polyketide synthase gene pks4 of Trichoderma reesei provides pigmentation and stress resistance. Eukaryot Cell, 12(11). https://doi.org/10.1128/EC.00103-13

Avilés, D., Espinoza, F., Villao, L., Alvarez, J., Sosa, D., Santos-Ordóñez, E., & Galarza, L. (2023). Application of microencapsulated Trichoderma spp. against Moniliophthora roreri during the vegetative development of cocoa. Scientia Agropecuaria, 14(4), 539-547. https://doi.org/10.17268/sci.agropecu.2023.045

Bae, Y. S., & Knudsen, G. R. (2000). Cotransformation of Trichoderma harzianum with β-glucuronidase and green fluorescent protein genes provides a useful tool for monitoring fungal growth and activity in natural soils. Appl Environ Microbiol, 66(2). https://doi.org/10.1128/AEM.66.2.810-815.2000

Cai, F., Kubicek, C. P., & Druzhinina, I. S. (2021). Genetic transformation of Trichoderma spp. In: Basu, C. (eds) Biofuels and Biodiesel. Methods in Molecular Biology, vol 2290. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1323-8_12

Çakmakçı, R., Salık, M. A., & Çakmakçı, S. (2023). Assessment and principles of environmentally sustainable food and agriculture systems. Agriculture, 13(5), 1073. https://doi.org/10.3390/agriculture13051073

Cardoza, R. E., Vizcaino, J. A., Hermosa, M. R., Monte, E., & Gutiérrez, S. (2006). A comparison of the phenotypic and genetic stability of recombinant Trichoderma spp. generated by protoplast - and Agrobacterium - mediated transformation. Journal of Microbiology, 44(4), 383-395.

Carvalho, E. A., dos Santos Góes, L. M., Uetanabaro, A. P. T., da Silva, E. G. P., Rodrigues, L. B., Pirovani, C. P., & da Costa, A. M. (2017). Thermoresistant xylanases from Trichoderma stromaticum: Application in bread making and manufacturing xylo-oligosaccharides. Food Chem, 221, 1499-1506. https://doi.org/10.1016/j.foodchem.2016.10.144

Chóez-Guaranda, I., Espinoza-Lozano, F., Reyes-Araujo, D., Romero, C., Manzano, P., Galarza, L., & Sosa, D. (2023). Chemical characterization of Trichoderma spp. extracts with antifungal activity against cocoa pathogens. Molecules, 28(7), 3208. https://doi.org/10.3390/molecules28073208

Chung, D., Kwon, Y. M., Lim, J. Y., Bae, S. S., Choi, G., & Lee, D. S. (2022). Characterization of chitinolytic and antifungal activities in marine-derived Trichoderma bissettii strains. Mycobiology, 50(4), 244-253. https://doi.org/10.1080/12298093.2022.2105509

Cortes, M. V. de C. B., Barbosa, E. T., Oliveira, M. I. de S., Maciel, L. H. R., Lobo Junior, M., et al. (2023). Trichoderma harzianum marker-free strain construction based on efficient CRISPR/Cas9 recyclable system: A helpful tool for the study of biological control agents. Biological Control, 184, 105281 https://doi.org/10.1016/j.biocontrol.2023.105281

De Paula, R. G., Antoniêto, A. C. C., Ribeiro, L. F. C., Carraro, C. B., Nogueira, K. M. V., et al. (2018). New genomic approaches to enhance biomass degradation by the industrial fungus Trichoderma reesei. Int J Genomics, 1974151. https://doi.org/10.1155/2018/1974151

Donzelli, B. G. G., Lorito, M., Scala, F., & Harman, G. E. (2001). Cloning, sequence and structure of a gene encoding an antifungal glucan 1,3-β-glucosidase from Trichoderma atroviride (T. harzianum). Gene, 277, 199-208. https://doi.org/10.1016/S0378-1119(01)00681-3

El-Saadony, M. T., Saad, A. M., Soliman, S. M., Salem, H. M., Ahmed, A. I., et al. (2022). Plant growth-promoting microorganisms as biocontrol agents of plant diseases: Mechanisms, challenges and future perspectives. Front Plant Sci, 13, 923880. https://doi.org/10.3389/fpls.2022.923880

Fazeli-Nasab, B., Shahraki-Mojahed, L., Piri, R., & Sobhanizadeh, A. (2022). Trichoderma: Improving growth and tolerance to biotic and abiotic stresses in plants, in: Trends of Applied Microbiology for Sustainable Economy. 525-564 https://doi.org/10.1016/B978-0-323-91595-3.00004-5

Ferreira Filho, J. A., Horta, M. A. C., dos Santos, C. A., Almeida, D. A., Murad, N. F., et al. (2020). Integrative genomic analysis of the bioprospection of regulators and accessory enzymes associated with cellulose degradation in a filamentous fungus (Trichoderma harzianum). BMC Genomics, 21, 757. https://doi.org/10.1186/s12864-020-07158-w

Fitz, E., Wanka, F., & Seiboth, B. (2018). The promoter toolbox for recombinant gene expression in Trichoderma reesei. Front Bioeng Biotechnol, 6, 135. https://doi.org/10.3389/FBIOE.2018.00135/BIBTEX

Galarza, L., Akagi, Y., Takao, K., Kim, C. S., Maekawa, N., et al. (2015). Characterization of Trichoderma species isolated in Ecuador and their antagonistic activities against phytopathogenic fungi from Ecuador and Japan. Journal of General Plant Pathology, 81, 201-210. https://doi.org/10.1007/S10327-015-0587-X/FIGURES/3

Gladek, E., Gerald, R., Muños Sabag, O., Kennedy, E., Fraser, M., & Hirsh, P. (2017). The Global Food System: An Analysis. Report commissioned by WWF Netherlands. Metabolic. https://www.metabolic.nl/publication/global-food-system-an-analysis/

Goldman, G. H., van Montagu, M., & Herrera-Estrella, A. (1990). Transformation of Trichoderma harzianum by high-voltage electric pulse. Curr Genet, 17, 169-174. https://doi.org/10.1007/BF00312863

Green, H., & Jensen, D. F. (1995). A tool for monitoring Trichoderma harzianum: II. The use of a GUS transformant for ecological studies in the rhizosphere. Phytopathology, 85, 1436-1440. https://doi.org/10.1094/Phyto-85-1436

Guo, Q., Shi, L., Wang, X., Li, D., Yin, Z., Zhang, J., Ding, G., & Chen, L. (2023). Structures and biological activities of secondary metabolites from the Trichoderma genus (Covering 2018-2022). J Agric Food Chem, 71(37), 13612-13632. https://doi.org/10.1021/acs.jafc.3c04540

Guzmán-Guzmán, P., Kumar, A., de los Santos-Villalobos, S., Parra-Cota, F. I., Orozco-Mosqueda, M. del C., et al. (2023). Trichoderma species: Our best fungal allies in the biocontrol of plant diseases - A Review. Plants, 12(3), 432 https://doi.org/10.3390/plants12030432

Hao, Z., & Su, X. (2019). Fast gene disruption in Trichoderma reesei using in vitro assembled Cas9/gRNA complex. BMC Biotechnol, 19, 2. https://doi.org/10.1186/s12896-018-0498-y

Harman, G. E., Howell, C. R., Viterbo, A., Chet, I., & Lorito, M. (2004). Trichoderma species - opportunistic, avirulent plant symbionts. Nat Rev Microbiol, 2, 43-56. https://doi.org/10.1038/NRMICRO797

Howell, C. R. (2003). Mechanisms employed by Trichoderma species in the biological control of plant diseases: The history and evolution of current concepts. Plant Disease, 87(1), 4-10. https://doi.org/10.1094/pdis.2003.87.1.4

Howell, C. R., Stipanovic, R. D., & Lumsden, R. D. (2008). Antibiotic production by strains of Gliocladium virens and its relation to the biocontrol of cotton seedling diseases. Biocontrol Science and Technology, 3(4), 435-441. https://doi.org/10.1080/09583159309355298

Iyyappan, J., Pravin, R., Al-Ghanim, K. A., Govindarajan, M., Nicoletti, M., & Baskar, G. (2023). Dual strategy for bioconversion of elephant grass biomass into fermentable sugars using Trichoderma reesei towards bioethanol production. Bioresour Technol, 374, 128804. https://doi.org/10.1016/j.biortech.2023.128804

Katayama, T., Tanaka, Y., Okabe, T., Nakamura, H., Fujii, W., Kitamoto, K., & Maruyama, J. (2016). Development of a genome editing technique using the CRISPR/Cas9 system in the industrial filamentous fungus Aspergillus oryzae. Biotechnol Lett, 38, 637-642. https://doi.org/10.1007/s10529-015-2015-x

Khan, R. A. A., Najeeb, S., Hussain, S., Xie, B., & Li, Y. (2020). Bioactive secondary metabolites from Trichoderma spp. Against phytopathogenic fungi. Microorganisms, 8(6), 817. https://doi.org/10.3390/microorganisms8060817

Kubicek, C. P., Steindorff, A. S., Chenthamara, K., Manganiello, G., Henrissat, B., Zhang, J., Cai, F., Kopchinskiy, A. G., Kubicek, E. M., Kuo, A., Baroncelli, R., Sarrocco, S., Noronha, E. F., Vannacci, G., Shen, Q., Grigoriev, I. V., & Druzhinina, I. S. (2019). Evolution and comparative genomics of the most common Trichoderma species. BMC Genomics, 20, 485 https://doi.org/10.1186/s12864-019-5680-7

Kück, U., & Hoff, B. (2010). New tools for the genetic manipulation of filamentous fungi. Appl Microbiol Biotechnol, 86, 51-62. https://doi.org/10.1007/s00253-009-2416-7

Li, D., Tang, Y., Lin, J., & Cai, W. (2017). Methods for genetic transformation of filamentous fungi. Microb Cell Fact, 16, 1-13. https://doi.org/10.1186/s12934-017-0785-7

Liu, D., Garrigues, S., & de Vries, R. P. (2023). Heterologous protein production in filamentous fungi. Appl Microbiol Biotechnol, 107, 5019-5033. https://doi.org/10.1007/s00253-023-12660-8

Liu, R., Chen, L., Jiang, Y., Zhou, Z., & Zou, G. (2015). Efficient genome editing in filamentous fungus Trichoderma reesei using the CRISPR/Cas9 system. Cell Discov, 1, 15007. https://doi.org/10.1038/celldisc.2015.7

Lorito, M., Hayes, C. K., di Pietro, A., & Harman, G. E. (1993). Biolistic transformation of Trichoderma harzianum and Gliocladium virens using plasmid and genomic DNA. Curr Genet, 24, 349-356. https://doi.org/10.1007/BF00336788

Magaña-Ortíz, D., Coconi-Linares, N., Ortiz-Vazquez, E., Fernández, F., Loske, A. M., & Gómez-Lim, M. A. (2013). A novel and highly efficient method for genetic transformation of fungi employing shock waves. Fungal Genetics and Biology, 56, 9-16. https://doi.org/10.1016/j.fgb.2013.03.008

Martín, J. F. (2015). Fungal transformation: From protoplasts to targeted recombination systems. In: van den Berg, M., Maruthachalam, K. (eds) Genetic Transformation Systems in Fungi, Volume 1. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-10142-2_1

Martinez, D., Berka, R. M., Henrissat, B., Saloheimo, M., Arvas, M., et al. (2008). Genome sequencing and analysis of the biomass-degrading fungus Trichoderma reesei (syn. Hypocrea jecorina). Nat Biotechnol, 26, 553-560. https://doi.org/10.1038/nbt1403

Maruyama, C. R., Bilesky-José, N., de Lima, R., & Fraceto, L. F. (2020). Encapsulation of Trichoderma harzianum preserves enzymatic activity and enhances the potential for biological control. Front Bioeng Biotechnol, 8, 225. https://doi.org/10.3389/fbioe.2020.00225

Mukherjee, P. K., Buensanteai, N., Moran-Diez, M. E., Druzhinina, I. S., & Kenerley, C. M. (2012a). Functional analysis of non-ribosomal peptide synthetases (NRPSs) in Trichoderma virens reveals a polyketide synthase (PKS)/NRPS hybrid enzyme involved in the induced systemic resistance response in maize. Microbiology, 158, 155-165. https://doi.org/10.1099/mic.0.052159-0

Mukherjee, P. K., Horwitz, B. A., Herrera-Estrella, A., Schmoll, M., & Kenerley, C. M. (2013). Trichoderma research in the Genome Era. Annual review of phytopathology, 51, 105-129. https://doi.org/10.1146/ANNUREV-PHYTO-082712-102353

Mukherjee, P. K., Horwitz, B. A., Kenerley, C. M. (2012b). Secondary metabolism in Trichoderma - A genomic perspective. Microbiology, 158, 35-45. https://doi.org/10.1099/mic.0.053629-0

Nogueira-López, G., Padilla-Arizmendi, F., Inwood, S., Lyne, S., Steyaert, J. M., Nieto-Jacobo, M. F., Stewart, A., & Mendoza-Mendoza, A. (2019). TrichoGate: An improved vector system for a large scale of functional analysis of Trichoderma genes. Front Microbiol, 10, 1-18. https://doi.org/10.3389/fmicb.2019.02794

Paloheimo, M., Haarmann, T., Mäkinen, S., & Vehmaanperä, J. (2016). Production of industrial enzymes in Trichoderma reesei. In: Schmoll, M., Dattenböck, C. (eds) Gene Expression Systems in Fungi: Advancements and Applications. Fungal Biology. Springer, Cham. https://doi.org/10.1007/978-3-319-27951-0_2

Pohl, C., Kiel, J. A. K. W., Driessen, A. J. M., Bovenberg, R. A. L., & Nygård, Y. (2016). CRISPR/Cas9 based genome editing of Penicillium chrysogenum. ACS Synth Biol, 5, 7, 754-764. https://doi.org/10.1021/acssynbio.6b00082

Qi, Q., Fan, C., Wu, H., Sun, L., & Cao, C. (2023). Preparation of Trichoderma asperellum microcapsules and biocontrol of cucumber powdery mildew. Microbiol Spectr, 11(3), e05084-22. https://doi.org/10.1128/spectrum.05084-22

Rantasalo, A., Vitikainen, M., Paasikallio, T., Jäntti, J., Landowski, C. P., & Mojzita, D. (2019). Novel genetic tools that enable highly pure protein production in Trichoderma reesei. Sci Rep, 9, 5032. https://doi.org/10.1038/s41598-019-41573-8

Rosmana, A., Taufik, M., Asman, A., Jayanti, N. J., & Hakkar, A. A. (2019). Dynamic of vascular streak dieback disease incidence on susceptible cacao treated with composted plant residues and Trichoderma asperellum in field. Agronomy, 9(10), 650. https://doi.org/10.3390/agronomy9100650

Rubio, M. B., Pardal, A. J., Cardoza, R. E., Gutiérrez, S., Monte, E., & Hermosa, R. (2017). Involvement of the transcriptional coactivator ThMBF1 in the biocontrol activity of Trichoderma harzianum. Front Microbiol, 8. https://doi.org/10.3389/fmicb.2017.02273

Sandle, T. (2014). Trichoderma. Encyclopedia of Food Microbiology: Second Edition, 644-646. https://doi.org/10.1016/B978-0-12-384730-0.00337-2

Saravanakumar, K., Rathnayake, A. U., Sathiyaseelan, A., Selvakumar, V., Vijaya Anand, M. A., et al. (2020). Trichoderma-functional metabolomics to genetic engineering. New and Future Developments in Microbial Biotechnology and Bioengineering, 209-220. https://doi.org/10.1016/B978-0-12-819453-9.00010-6

Schalamun, M., & Schmoll, M. (2022). Trichoderma - genomes and genomics as treasure troves for research towards biology, biotechnology and agriculture. Frontiers in Fungal Biology, 3. https://doi.org/10.3389/ffunb.2022.1002161

Schmoll, M., & Schuster, A. (2010). Biology and biotechnology of Trichoderma. Appl Microbiol Biotechnol, 87, 787-799. https://doi.org/10.1007/s00253-010-2632-1

Schuster, M., Schweizer, G., Reissmann, S., & Kahmann, R. (2016). Genome editing in Ustilago maydis using the CRISPR-Cas system. Fungal Genetics and Biology, 89, 3-9. https://doi.org/10.1016/j.fgb.2015.09.001

Shi, T. Q., Liu, G. N., Ji, R. Y., Shi, K., Song, P., Ren, L. J., Huang, H., & Ji, X. J. (2017). CRISPR/Cas9-based genome editing of the filamentous fungi: the state of the art. Appl Microbiol Biotechnol, 101, 7435-7443. https://doi.org/10.1007/s00253-017-8497-9

Simamora, M., Basyuni, M., & Lisnawita, (2021). Potency of secondary metabolites of Trichoderma asperellum and Pseudomonas fluorescens in the growth of cocoa plants affected by vascular streak dieback. Biodiversitas, 22(5), 2542-2547. https://doi.org/10.13057/biodiv/d220511

Steen, E. J., Kang, Y., Bokinsky, G., Hu, Z., Schirmer, A., McClure, A., del Cardayre, S. B., & Keasling, J. D. (2010). Microbial production of fatty-acid-derived fuels and chemicals from plant biomass. Nature, 463, 559-562. https://doi.org/10.1038/nature08721

Stoppacher, N., Kluger, B., Zeilinger, S., Krska, R., & Schuhmacher, R. (2010). Identification and profiling of volatile metabolites of the biocontrol fungus Trichoderma atroviride by HS-SPME-GC-MS. J Microbiol Methods, 81(2), 187-193. https://doi.org/10.1016/j.mimet.2010.03.011

Stracquadanio, C., Quiles, J. M., Meca, G., & Cacciola, S. O. (2020). Antifungal activity of bioactive metabolites produced by Trichoderma asperellum and Trichoderma atroviride in liquid medium. Journal of Fungi, 6(4), 263. https://doi.org/10.3390/jof6040263

Suarez, B., Rey, M., Castillo, P., Monte, E., & Llobell, A. (2004). Isolation and characterization of PRA1, a trypsin-like protease from the biocontrol agent Trichoderma harzianum CECT 2413 displaying nematicidal activity. Appl Microbiol Biotechnol, 65, 46-55. https://doi.org/10.1007/S00253-004-1610-X

Szabó, M., Urbán, P., Virányi, F., Kredics, L., & Fekete, C. (2013). Comparative gene expression profiles of Trichoderma harzianum proteases during in vitro nematode egg-parasitism. Biological Control, 67, 337-343. https://doi.org/10.1016/J.BIOCONTROL.2013.09.002

Tyśkiewicz, R., Nowak, A., Ozimek, E., & Jaroszuk-ściseł, J. (2022). Trichoderma: The current status of its application in agriculture for the biocontrol of fungal phytopathogens and stimulation of plant growth. Int J Mol Sci, 23(4), 2329. https://doi.org/10.3390/ijms23042329

Valenzuela-Cobos, J. D., Guevara-Viejó, F., Vicente-Galindo, P., & Galindo-Villardón, P. (2023). Eco-Friendly Biocontrol of Moniliasis in Ecuadorian Cocoa Using Biplot Techniques. Sustainability, 15(5), 4223. https://doi.org/10.3390/su15054223

Vieira, A. A., Vianna, G. R., Carrijo, J., Aragão, F. J. L., & Vieira, P. M. (2021). Generation of Trichoderma harzianum with pyr4 auxotrophic marker by using the CRISPR/Cas9 system. Sci Rep, 11, 1085. https://doi.org/10.1038/s41598-020-80186-4

Wang, Q., Chen, L., Fang, C., Wang, H., Shi, Y., & Zhao, Y. (2019). The overexpression of one single cbh gene making Trichoderma asperellum T-1 a better cellulase producer. Ann Microbiol, 69, 673-683. https://doi.org/10.1007/s13213-019-01458-7

Wang, W., Shi, X. Y., & Wei, D. Z. (2014). Light-mediated control of gene expression in filamentous fungus Trichoderma reesei. J Microbiol Methods, 103, 37-39. https://doi.org/10.1016/j.mimet.2014.05.017

Wang, Y., Chen, H., Ma, L., Gong, M., Wu, Y., Bao, D., & Zou, G. (2022). Use of CRISPR-Cas tools to engineer Trichoderma species. Microb Biotechnol, 15, 2521-2532. https://doi.org/10.1111/1751-7915.14126

Wiest, A., Grzegorski, D., Xu, B. W., Goulard, C., Rebuffat, S., Ebbole, D. J., Bodo, B., & Kenerley, C. (2002). Identification of peptaibols from Trichoderma virens and cloning of a peptaibol synthetase. Journal of Biological Chemistry, 277(23), 20862-20868. https://doi.org/10.1074/jbc.M201654200

Wilson, R. A., & Talbot, N. J. (2009). Fungal physiology - A future perspective. Microbiology, 155(12), 3810-3815. https://doi.org/10.1099/mic.0.035436-0

Wu, C., Chen, Y., Huang, X., Sun, S., Luo, J., Lu, Z., Wang, W., & Ma, Y. (2019). An efficient shortened genetic transformation strategy for filamentous fungus Trichoderma reesei. Journal of General and Applied Microbiology, 65(6), 301-307. https://doi.org/10.2323/jgam.2019.02.001

Wu, C., Chen, Y., Qiu, Y., Niu, X., Zhu, N., Chen, J., Yao, H., Wang, W., & Ma, Y. (2020). A simple approach to mediate genome editing in the filamentous fungus Trichoderma reesei by CRISPR/Cas9-coupled in vivo gRNA transcription. Biotechnol Lett, 42, 1203-1210. https://doi.org/10.1007/s10529-020-02887-0

Xu, Q., Himmel, M. E., & Singh, A. (2015). Production of ethanol from engineered Trichoderma reesei. Direct microbial conversion of biomass to advanced biofuels, 197-208. https://doi.org/10.1016/B978-0-444-59592-8.00011-7

Yao, L., Tan, C., Song, J., Yang, Q., Yu, L., & Li, X. (2016). Isolation and expression of two polyketide synthase genes from Trichoderma harzianum 88 during mycoparasitism. Brazilian Journal of Microbiology, 47(2), 468-479. https://doi.org/10.1016/j.bjm.2016.01.004

Yao, X., Guo, H., Zhang, K., Zhao, M., Ruan, J., & Chen, J. (2023). Trichoderma and its role in biological control of plant fungal and nematode disease. Front Microbiol, 14. https://doi.org/10.3389/fmicb.2023.1160551

Yazawa, H., Kumagai, H., & Uemura, H. (2013). Secretory production of ricinoleic acid in fission yeast Schizosaccharomyces pombe. Appl Microbiol Biotechnol, 97, 8663-8671. https://doi.org/10.1007/s00253-013-5060-1

Zeilinger, S. (2004). Gene disruption in Trichoderma atroviride via Agrobacterium-mediated transformation. Curr Genet, 45. https://doi.org/10.1007/s00294-003-0454-8

Zeilinger, S., Gruber, S., Bansal, R., & Mukherjee, P. K. (2016). Secondary metabolism in Trichoderma - Chemistry meets genomics. Fungal Biol Rev, 30(2), 74-90. https://doi.org/10.1016/j.fbr.2016.05.001

Zhang, C., Meng, X., Wei, X., & Lu, L. (2016). Highly efficient CRISPR mutagenesis by microhomology-mediated end joining in Aspergillus fumigatus. Fungal Genetics and Biology, 86. https://doi.org/10.1016/j.fgb.2015.12.007

Zhang, J. L., Tang, W. L., Huang, Q. R., Li, Y. Z., Wei, M. L., et al. (2021). Trichoderma: A treasure house of structurally diverse secondary metabolites with medicinal importance. Front Microbiol, 12. https://doi.org/10.3389/fmicb.2021.723828

Zhang, L., Zhao, X., Zhang, G., Zhang, J., Wang, X., Zhang, S., Wang, W., & Wei, D. (2016). Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0. Sci Rep, 6, 20761. https://doi.org/10.1038/srep20761

Zhang, Y., Adams, I. P., & Ratledge, C. (2007) Malic enzyme: the controlling activity for lipid production? Overexpression of malic enzyme in Mucor circinelloides leads to a 2.5-fold increase in lipid accumulation. Microbiology, 153, 2013-2025. https://doi.org/10.1099/MIC.0.2006/002683-0

Zin, N., & Badaluddin, N. (2020). Biological functions of Trichoderma spp. for agriculture applications. Annals of Agricultural Sciences, 65(2), 168-178. https://doi.org/10.1016/j.aoas.2020.09.003