Exploration of Trichoderma reesei as an alternative host for erythritol production.

BACKGROUND: Erythritol, a natural polyol, is a low-calorie sweetener synthesized by a number of microorganisms, such as Moniliella pollinis. Yet, a widespread use of erythritol is limited by high production costs due to the need for cultivation on glucose-rich substrates. This study explores the potential of using Trichoderma reesei as an alternative host for erythritol production, as this saprotrophic fungus can be cultivated on lignocellulosic biomass residues. The objective of this study was to evaluate whether such an alternative host would lead to a more sustainable and economically viable production of erythritol by identifying suitable carbon sources for erythritol biosynthesis, the main parameters influencing erythritol biosynthesis and evaluating the feasibility of scaling up the defined process. RESULTS: Our investigation revealed that T. reesei can synthesize erythritol from glucose but not from other carbon sources like xylose and lactose. T. reesei is able to consume erythritol, but it does not in the presence of glucose. Among nitrogen sources, urea and yeast extract were more effective than ammonium and nitrate. A significant impact on erythritol synthesis was observed with variations in pH and temperature. Despite successful shake flask experiments, the transition to bioreactors faced challenges, indicating a need for further scale-up optimization. CONCLUSIONS: While T. reesei shows potential for erythritol production, reaching a maximum concentration of 1 g/L over an extended period, its productivity could be improved by optimizing the parameters that affect erythritol production. In any case, this research contributes valuable insights into the polyol metabolism of T. reesei, offering potential implications for future research on glycerol or mannitol production. Moreover, it suggests a potential metabolic association between erythritol production and glycolysis over the pentose phosphate pathway.

Bi-directionalized promoter systems allow methanol-free production of hard-to-express peroxygenases with Komagataella Phaffii.

BACKGROUND: Heme-incorporating peroxygenases are responsible for electron transport in a multitude of organisms. Yet their application in biocatalysis is hindered due to their challenging recombinant production. Previous studies suggest Komagataella phaffi to be a suitable production host for heme-containing enzymes. In addition, co-expression of helper proteins has been shown to aid protein folding in yeast. In order to facilitate recombinant protein expression for an unspecific peroxygenase (AnoUPO), we aimed to apply a bi-directionalized expression strategy with Komagataella phaffii. RESULTS: In initial screenings, co-expression of protein disulfide isomerase was found to aid the correct folding of the expressed unspecific peroxygenase in K. phaffi. A multitude of different bi-directionalized promoter combinations was screened. The clone with the most promising promoter combination was scaled up to bioreactor cultivations and compared to a mono-directional construct (expressing only the peroxygenase). The strains were screened for the target enzyme productivity in a dynamic matter, investigating both derepression and mixed feeding (methanol-glycerol) for induction. Set-points from bioreactor screenings, resulting in the highest peroxygenase productivity, for derepressed and methanol-based induction were chosen to conduct dedicated peroxygenase production runs and were analyzed with RT-qPCR. Results demonstrated that methanol-free cultivation is superior over mixed feeding in regard to cell-specific enzyme productivity. RT-qPCR analysis confirmed that mixed feeding resulted in high stress for the host cells, impeding high productivity. Moreover, the bi-directionalized construct resulted in a much higher specific enzymatic activity over the mono-directional expression system. CONCLUSIONS: In this study, we demonstrate a methanol-free bioreactor production strategy for an unspecific peroxygenase, yet not shown in literature. Hence, bi-directionalized assisted protein expression in K. phaffii, cultivated under derepressed conditions, is indicated to be an effective production strategy for heme-containing oxidoreductases. This very production strategy might be opening up further opportunities for biocatalysis.

Genomic deletions in Aureobasidium pullulans by an AMA1 plasmid for gRNA and CRISPR/Cas9 expression.

BACKGROUND: Aureobasidium pullulans is a generalist polyextremotolerant black yeast fungus. It tolerates temperatures below 0 °C or salt concentrations up to 18%, among other stresses. A. pullulans genome sequencing revealed a high potential for producing bioactive metabolites. Only few molecular tools exist to edit the genome of A. pullulans, hence it is important to make full use of its potential. Two CRISPR/Cas9 methods have been proposed for the protoplast-based transformation of A. pullulans. These methods require the integration of a marker gene into the locus of the gene to be deleted, when the deletion of this gene does not yield a selectable phenotype. We present the adaptation of a plasmid-based CRISPR/Cas9 system developed in Aspergillus niger for A. pullulans to create deletion strains. RESULTS: The A. niger CRISPR/Cas9 plasmid led to efficient genomic deletions in A. pullulans. In this study, strains with deletions ranging from 30 to 862 bp were obtained by using an AMA1 plasmid-based genome editing strategy. CONCLUSION: The CRISPR/Cas9 transformation system presented in this study provides new opportunities for strain engineering of A. pullulans. This system allows expression of Cas9 and antibiotic resistance while being easy to adapt. This strategy could open the path to intensive genomic engineering in A. pullulans.

Protein secretion and associated stress in industrially employed filamentous fungi.

Application of filamentous fungi for the production of commercial enzymes such as amylase, cellulase, or xylanase is on the rise due to the increasing demand to degrade several complex carbohydrates as raw material for biotechnological processes. Also, protein production by fungi for food and feed gains importance. In any case, the protein production involves both cellular synthesis and secretion outside of the cell. Unfortunately, the secretion of proteins or enzymes can be hampered due to accumulation of unfolded or misfolded proteins in the endoplasmic reticulum (ER) as a result of too high synthesis of enzymes or (heterologous) protein expression. To cope with this ER stress, the cell generates a response known as unfolded protein response (UPR). Even though this mechanism should re-establish the protein homeostasis equivalent to a cell under non-stress conditions, the enzyme expression might still suffer from repression under secretory stress (RESS). Among eukaryotes, Saccharomyces cerevisiae is the only fungus, which is studied quite extensively to unravel the UPR pathway. Several homologs of the proteins involved in this signal transduction cascade are also found in filamentous fungi. Since RESS seems to be absent in S. cerevisiae and was only reported in Trichoderma reesei in the presence of folding and glycosylation inhibitors such as dithiothreitol and tunicamycin, more in-depth study about this mechanism, specifically in filamentous fungi, is the need of the hour. Hence, this review article gives an overview on both, protein secretion and associated stress responses in fungi. KEY POINTS: • Enzymes produced by filamentous fungi are crucial in industrial processes • UPR mechanism is conserved among many fungi, but mediated by different proteins • RESS is not fully understood or studied in industrially relevant filamentous fungi.

The phenomenon of strain degeneration in biotechnologically relevant fungi.

Fungi are widely exploited for large-scale production in the biotechnological industry to produce a diverse range of substances due to their versatility and relative ease of growing on various substrates. The occurrence of a phenomenon-the so-called fungal strain degeneration-leads to the spontaneous loss or decline of production capacity and results in an economic loss on a tremendous scale. Some of the most commonly applied genera of fungi in the biotechnical industry, such as Aspergillus, Trichoderma, and Penicillium, are threatened by this phenomenon. Although fungal degeneration has been known for almost a century, the phenomenon and its underlying mechanisms still need to be understood. The proposed mechanisms causing fungi to degenerate can be of genetic or epigenetic origin. Other factors, such as culture conditions, stress, or aging, were also reported to have an influence. This mini-review addresses the topic of fungal degeneration by describing examples of productivity losses in biotechnical processes using Aspergillus niger, Aspergillus oryzae, Trichoderma reesei, and Penicillium chrysogenum. Further, potential reasons, circumvention, and prevention methods are discussed. This is the first mini-review which provides a comprehensive overview on this phenomenon in biotechnologically used fungi, and it also includes a collection of strategies that can be useful to minimize economic losses which can arise from strain degeneration. KEY POINTS: • Spontaneous loss of productivity is evident in many fungi used in biotechnology. • The properties and mechanisms underlying this phenomenon are very versatile. • Only studying these underlying mechanisms enables the design of a tailored solution.

Evaluation of reference genes for transcript analyses in Komagataella phaffii (Pichia pastoris).

BACKGROUND: The yeast Komagataella phaffii (Pichia pastoris) is routinely used for heterologous protein expression and is suggested as a model organism for yeast. Despite its importance and application potential, no reference gene for transcript analysis via RT-qPCR assays has been evaluated to date. In this study, we searched publicly available RNASeq data for stably expressed genes to find potential reference genes for relative transcript analysis by RT-qPCR in K. phaffii. To evaluate the applicability of these genes, we used a diverse set of samples from three different strains and a broad range of cultivation conditions. The transcript levels of 9 genes were measured and compared using commonly applied bioinformatic tools. RESULTS: We could demonstrate that the often-used reference gene ACT1 is not very stably expressed and could identify two genes with outstandingly low transcript level fluctuations. Consequently, we suggest the two genes, RSC1, and TAF10 to be simultaneously used as reference genes in transcript analyses by RT-qPCR in K. phaffii in future RT-qPCR assays. CONCLUSION: The usage of ACT1 as a reference gene in RT-qPCR analysis might lead to distorted results due to the instability of its transcript levels. In this study, we evaluated the transcript levels of several genes and found RSC1 and TAF10 to be extremely stable. Using these genes holds the promise for reliable RT-qPCR results.

In Vitro Characterization of a Nuclear Receptor-like Domain of the Xylanase Regulator 1 from Trichoderma reesei.

Engineering transcription factors is an interesting research target gaining increasing attention, such as in the case of industrially used organisms. With respect to sustainability, biomass-degrading saprophytic fungi, such as Trichoderma reesei, are promising industrial work horses because they exhibit a high secretory capacity of native and heterologously expressed enzymes and compounds. A single-point mutation in the main transactivator of xylanase and cellulase expressions in T. reesei Xyr1 led to a strongly deregulated and enhanced xylanase expression. Circular dichroism spectroscopy revealed a change in secondary structure caused by this mutation. According to electrophoretic mobility shift assays and determination of the equilibrium-binding constants, the DNA-binding affinity of the mutated Xyr1 was considerably reduced compared to the wild-type Xyr1. Both techniques were also used to investigate the allosteric response to carbohydrates (D-glucose-6-phosphate, D-xylose, and sophorose) signalling the repression or induction of Xyr1 target genes. The mutated Xyr1 no longer exhibited a conformational change in response to these carbohydrates, indicating that the observed deregulation is not a simple matter of a change in DNA-binding of the transactivator. Altogether, we postulate that the part of Xyr1 where the mutation is located functions as a nuclear receptor-like domain that mediates carbohydrate signals and modulates the Xyr1 transactivating activity.

A bioinformatic-assisted workflow for genome-wide identification of ncRNAs.

With the upcoming of affordable Next-Generation Sequencing technologies, the number of known non-protein coding RNAs increased drastically in recent years. Different types of non-coding RNAs (ncRNAs) emerged as key players in the regulation of gene expression on the RNA-RNA, RNA-DNA as well as RNA-protein level, ranging from involvement in chromatin remodeling and transcription regulation to post-transcriptional modifications. Prediction of ncRNAs involves the use of several bioinformatics tools and can be a daunting task for researchers. This led to the development of analysis pipelines such as UClncR and lncpipe. However, these pipelines are limited to datasets from human, mouse, zebrafish or fruit fly and are not able to analyze RNA sequencing data from other organisms. In this study, we developed the analysis pipeline Pinc (Pipeline for prediction of ncRNA) as an enhanced tool to predict ncRNAs based on sequencing data by removing transcripts that show protein-coding potential. Additionally, a feature for differential expression analysis of annotated genes as well as for identification of novel ncRNAs is implemented. Pinc uses Nextflow as a framework and is built with robust and well-established analysis tools. This will allow researchers to utilize sequencing data from every organism in order to reliably identify ncRNAs.

Fast and efficient CRISPR-mediated genome editing in Aureobasidium using Cas9 ribonucleoproteins.

Species of the genus Aureobasidium are ubiquitous, polyextremotolerant, "yeast-like" ascomycetes used for the industrial production of pullulan and other products and as biocontrol agents in agriculture. Their application potential and wide-spread occurrence make Aureobasidium spp. interesting study objects. The availability of a fast and efficient genome editing method is an obvious advantage for future basic and applied research on Aureobasidium. In this study, we describe the development of a CRISPR/Cas9-based genome editing method using ribonucleoproteins (RNPs) in A. pullulans and A. melanogenum. We demonstrate that this method can be used for single and multiplex genome editing using only RNPs by targeting URA3 (encoding for orotidine-5'-phosphate decarboxylase), ADE2 (encoding for phosphoribosylaminoimidazole carboxylase) and ARG4 (encoding for argininosuccinate lyase). We demonstrate the applicability of Trichoderma reesei pyr4 and Aspergillus fumigatus pyrG to complement the URA3 deficiency. Further, we show that using RNPs improves the homologous recombination rate and 20 bp long homologous flanks are sufficient. Therefore, the repair cassettes can be constructed by a single PCR, abolishing the need for laborious and time-consuming cloning, which is necessary for previously described methods for CRISPR-mediated genome editing in these fungi. The here presented method allows fast and efficient genome editing for gene deletions, modifications, and insertions in Auresobasidium with a minimized risk of off-target effects.

FunOrder 2.0 - a method for the fully automated curation of co-evolved genes in fungal biosynthetic gene clusters.

Coevolution is an important biological process that shapes interacting proteins - may it be physically interacting proteins or consecutive enzymes in a metabolic pathway, such as the biosynthetic pathways for secondary metabolites. Previously, we developed FunOrder, a semi-automated method for the detection of co-evolved genes, and demonstrated that FunOrder can be used to identify essential genes in biosynthetic gene clusters from different ascomycetes. A major drawback of this original method was the need for a manual assessment, which may create a user bias and prevents a high-throughput application. Here we present a fully automated version of this method termed FunOrder 2.0. In the improved version, we use several mathematical indices to determine the optimal number of clusters in the FunOrder output, and a subsequent k-means clustering based on the first three principal components of a principal component analysis of the FunOrder output to automatically detect co-evolved genes. Further, we replaced the BLAST tool with the DIAMOND tool as a prerequisite for using larger proteome databases. Potentially, FunOrder 2.0 may be used for the assessment of complete genomes, which has not been attempted yet. However, the introduced changes slightly decreased the sensitivity of this method, which is outweighed by enhanced overall speed and specificity.

An overview on current molecular tools for heterologous gene expression in Trichoderma.

Fungi of the genus Trichoderma are routinely used as biocontrol agents and for the production of industrial enzymes. Trichoderma spp. are interesting hosts for heterologous gene expression because their saprotrophic and mycoparasitic lifestyles enable them to thrive on a large number of nutrient sources and some members of this genus are generally recognized as safe (GRAS status). In this review, we summarize and discuss several aspects involved in heterologous gene expression in Trichoderma, including transformation methods, genome editing strategies, native and synthetic expression systems and implications of protein secretion. This review focuses on the industrial workhorse Trichoderma reesei because this fungus is the best-studied member of this genus for protein expression and secretion. However, the discussed strategies and tools can be expected to be transferable to other Trichoderma species.

FunOrder: A robust and semi-automated method for the identification of essential biosynthetic genes through computational molecular co-evolution.

Secondary metabolites (SMs) are a vast group of compounds with different structures and properties that have been utilized as drugs, food additives, dyes, and as monomers for novel plastics. In many cases, the biosynthesis of SMs is catalysed by enzymes whose corresponding genes are co-localized in the genome in biosynthetic gene clusters (BGCs). Notably, BGCs may contain so-called gap genes, that are not involved in the biosynthesis of the SM. Current genome mining tools can identify BGCs, but they have problems with distinguishing essential genes from gap genes. This can and must be done by expensive, laborious, and time-consuming comparative genomic approaches or transcriptome analyses. In this study, we developed a method that allows semi-automated identification of essential genes in a BGC based on co-evolution analysis. To this end, the protein sequences of a BGC are blasted against a suitable proteome database. For each protein, a phylogenetic tree is created. The trees are compared by treeKO to detect co-evolution. The results of this comparison are visualized in different output formats, which are compared visually. Our results suggest that co-evolution is commonly occurring within BGCs, albeit not all, and that especially those genes that encode for enzymes of the biosynthetic pathway are co-evolutionary linked and can be identified with FunOrder. In light of the growing number of genomic data available, this will contribute to the studies of BGCs in native hosts and facilitate heterologous expression in other organisms with the aim of the discovery of novel SMs.

The pentose phosphate pathway in industrially relevant fungi: crucial insights for bioprocessing.

The pentose phosphate pathway (PPP) is one of the most targeted pathways in metabolic engineering. This pathway is the primary source of NADPH, and it contributes in fungi to the production of many compounds of interest such as polyols, biofuels, carotenoids, or antibiotics. However, the regulatory mechanisms of the PPP are still not fully known. This review provides an insight into the current comprehension of the PPP in fungi and the limitations of this current understanding. It highlights how this knowledge contributes to targeted engineering of the PPP and thus to better performance of industrially used fungal strains. KEY POINTS: • Type of carbon and nitrogen source as well as oxidative stress influence the PPP. • A complex network of transcription factors regulates the PPP. • Improved understanding of the PPP will allow to increase yields of bioprocesses.

Genome Sequence of the Black Yeast-Like Strain Aureobasidium pullulans var. aubasidani CBS 100524.

In this work, we present the whole-genome sequence and the complete mitochondrial sequence of the black yeast-like strain Aureobasidium pullulans var. aubasidani CBS 100524, which produces the exopolysaccharide aubasidan and was previously isolated from Betula sp. slime flux from the Leningrad Region of Russia.

The Potential of Synthetic Biology for Trichoderma reesei.

Within the last 20 years, ground-breaking progress has been made in the field of synthetic biology, enabling the construction of novel pathways up to entire synthetic genomes in both prokaryotic and eukaryotic organisms. These innovations are primarily adapted for biotechnological applications, where filamentous fungi such as Trichoderma reesei are widely used to produce various enzymes of industrial interest. In the following chapter, we provide a broad overview on the current progress involving this particular organism, covering studies on synthetic promoters and transcription factors as well as synthetic expression platforms. Furthermore, this chapters aims to be a short introduction to the present book since many methods mentioned here are described in detail in the subsequent chapters.

Nanoscale Infrared Spectroscopy and Chemometrics Enable Detection of Intracellular Protein Distribution.

Determination of the intracellular location of proteins is one of the fundamental tasks of microbiology. Conventionally, label-based microscopy and super-resolution techniques are employed. In this work, we demonstrate a new technique that can determine intracellular protein distribution at nanometer spatial resolution. This method combines nanoscale spatial resolution chemical imaging using the photothermal-induced resonance (PTIR) technique with multivariate modeling to reveal the intracellular distribution of cell components. Here, we demonstrate its viability by imaging the distribution of major cellulases and xylanases in Trichoderma reesei using the colocation of a fluorescent label (enhanced yellow fluorescence protein, EYFP) with the target enzymes to calibrate the chemometric model. The obtained partial least squares model successfully shows the distribution of these proteins inside the cell and opens the door for further studies on protein secretion mechanisms using PTIR.

Novel approach in whole genome mining and transcriptome analysis reveal conserved RiPPs in Trichoderma spp.

BACKGROUND: Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a highly diverse group of secondary metabolites (SM) of bacterial and fungal origin. While RiPPs have been intensively studied in bacteria, little is known about fungal RiPPs. In Fungi only six classes of RiPPs are described. Current strategies for genome mining are based on these six known classes. However, the genes involved in the biosynthesis of theses RiPPs are normally organized in biosynthetic gene clusters (BGC) in fungi. RESULTS: Here we describe a comprehensive strategy to mine fungal genomes for RiPPs by combining and adapting existing tools (e.g. antiSMASH and RiPPMiner) followed by extensive manual curation based on conserved domain identification, (comparative) phylogenetic analysis, and RNASeq data. Deploying this strategy, we could successfully rediscover already known fungal RiPPs. Further, we analysed four fungal genomes from the Trichoderma genus. We were able to find novel potential RiPP BGCs in Trichoderma using our unconventional mining approach. CONCLUSION: We demonstrate that the unusual mining approach using tools developed for bacteria can be used in fungi, when carefully curated. Our study is the first report of the potential of Trichoderma to produce RiPPs, the detected clusters encode novel uncharacterized RiPPs. The method described in our study will lead to further mining efforts in all subdivisions of the fungal kingdom.

Regulatory systems for gene expression control in cyanobacteria.

As photosynthetic microbes, cyanobacteria are attractive hosts for the production of high-value molecules from CO2 and light. Strategies for genetic engineering and tightly controlled gene expression are essential for the biotechnological application of these organisms. Numerous heterologous or native promoter systems were used for constitutive and inducible expression, yet many of them suffer either from leakiness or from a low expression output. Anyway, in recent years, existing systems have been improved and new promoters have been discovered or engineered for cyanobacteria. Moreover, alternative tools and strategies for expression control such as riboswitches, riboregulators or genetic circuits have been developed. In this mini-review, we provide a broad overview on the different tools and approaches for the regulation of gene expression in cyanobacteria and explain their advantages and disadvantages.

Regulation of gene expression by the action of a fungal lncRNA on a transactivator.

Long non-coding RNAs (lncRNAs) are crucial factors acting on regulatory processes in eukaryotes. Recently, for the first time in a filamentous fungus, the lncRNA HAX1 was characterized in the ascomycete Trichoderma reesei. In industry, this fungus is widely applied for the high-yield production of cellulases. The lncRNA HAX1 was reported to influence the expression of cellulase-encoding genes; interestingly, this effect is dependent on the presence of its most abundant length. Clearly, HAX1 acts in association with a set of well-described transcription factors to regulate gene expression. In this study, we attempted to elucidate the regulatory strategy of HAX1 and its interactions with the major transcriptional activator Xylanase regulator 1 (Xyr1). We demonstrated that HAX1 interferes with the negative feedback regulatory loop of Xyr1 in a sophisticated manner and thus ultimately has a positive effect on gene expression.