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.

A quantitative metabolic analysis reveals Acetobacterium woodii as a flexible and robust host for formate-based bioproduction.

Cheap and renewable feedstocks such as the one-carbon substrate formate are emerging for sustainable production in a growing chemical industry. We investigated the acetogen Acetobacterium woodii as a potential host for bioproduction from formate alone and together with autotrophic and heterotrophic co-substrates by quantitatively analyzing physiology, transcriptome, and proteome in chemostat cultivations in combination with computational analyses. Continuous cultivations with a specific growth rate of 0.05 h-1 on formate showed high specific substrate uptake rates (47 mmol g-1 h-1). Co-utilization of formate with H2, CO, CO2 or fructose was achieved without catabolite repression and with acetate as the sole metabolic product. A transcriptomic comparison of all growth conditions revealed a distinct adaptation of A. woodii to growth on formate as 570 genes were changed in their transcript level. Transcriptome and proteome showed higher expression of the Wood-Ljungdahl pathway during growth on formate and gaseous substrates, underlining its function during utilization of one-carbon substrates. Flux balance analysis showed varying flux levels for the WLP (0.7-16.4 mmol g-1 h-1) and major differences in redox and energy metabolism. Growth on formate, H2/CO2, and formate + H2/CO2 resulted in low energy availability (0.20-0.22 ATP/acetate) which was increased during co-utilization with CO or fructose (0.31 ATP/acetate for formate + H2/CO/CO2, 0.75 ATP/acetate for formate + fructose). Unitrophic and mixotrophic conversion of all substrates was further characterized by high energetic efficiencies. In silico analysis of bioproduction of ethanol and lactate from formate and autotrophic and heterotrophic co-substrates showed promising energetic efficiencies (70-92%). Collectively, our findings reveal A. woodii as a promising host for flexible and simultaneous bioconversion of multiple substrates, underline the potential of substrate co-utilization to improve the energy availability of acetogens and encourage metabolic engineering of acetogenic bacteria for the efficient synthesis of bulk chemicals and fuels from sustainable one carbon substrates.

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.

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.

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.

Transcription factor Xpp1 is a switch between primary and secondary fungal metabolism.

Fungi can produce a wide range of chemical compounds via secondary metabolism. These compounds are of major interest because of their (potential) application in medicine and biotechnology and as a potential source for new therapeutic agents and drug leads. However, under laboratory conditions, most secondary metabolism genes remain silent. This circumstance is an obstacle for the production of known metabolites and the discovery of new secondary metabolites. In this study, we describe the dual role of the transcription factor Xylanase promoter binding protein 1 (Xpp1) in the regulation of both primary and secondary metabolism of Trichoderma reesei Xpp1 was previously described as a repressor of xylanases. Here, we provide data from an RNA-sequencing analysis suggesting that Xpp1 is an activator of primary metabolism. This finding is supported by our results from a Biolog assay determining the carbon source assimilation behavior of an xpp1 deletion strain. Furthermore, the role of Xpp1 as a repressor of secondary metabolism is shown by gene expression analyses of polyketide synthases and the determination of the secondary metabolites of xpp1 deletion and overexpression strains using an untargeted metabolomics approach. The deletion of Xpp1 resulted in the enhanced secretion of secondary metabolites in terms of diversity and quantity. Homologs of Xpp1 are found among a broad range of fungi, including the biocontrol agent Trichoderma atroviride, the plant pathogens Fusarium graminearum and Colletotrichum graminicola, the model organism Neurospora crassa, the human pathogen Sporothrix schenckii, and the ergot fungus Claviceps purpurea.

The Viral Gene ORF79 Encodes a Repressor Regulating Induction of the Lytic Life Cycle in the Haloalkaliphilic Virus ϕCh1.

In this study, we describe the construction of the first genetically modified mutant of a halovirus infecting haloalkaliphilic Archaea By random choice, we targeted ORF79, a currently uncharacterized viral gene of the haloalkaliphilic virus ϕCh1. We used a polyethylene glycol (PEG)-mediated transformation method to deliver a disruption cassette into a lysogenic strain of the haloalkaliphilic archaeon Natrialba magadii bearing ϕCh1 as a provirus. This approach yielded mutant virus particles carrying a disrupted version of ORF79. Disruption of ORF79 did not influence morphology of the mature virions. The mutant virus was able to infect cured strains of N. magadii, resulting in a lysogenic, ORF79-disrupted strain. Analysis of this strain carrying the mutant virus revealed a repressor function of ORF79. In the absence of gp79, onset of lysis and expression of viral proteins occurred prematurely compared to their timing in the wild-type strain. Constitutive expression of ORF79 in a cured strain of N. magadii reduced the plating efficiency of ϕCh1 by seven orders of magnitude. Overexpression of ORF79 in a lysogenic strain of N. magadii resulted in an inhibition of lysis and total absence of viral proteins as well as viral progeny. In further experiments, gp79 directly regulated the expression of the tail fiber protein ORF34 but did not influence the methyltransferase gene ORF94. Further, we describe the establishment of an inducible promoter for in vivo studies in N. magadiiIMPORTANCE Genetic analyses of haloalkaliphilic Archaea or haloviruses are only rarely reported. Therefore, only little insight into the in vivo roles of proteins and their functions has been gained so far. We used a reverse genetics approach to identify the function of a yet undescribed gene of ϕCh1. We provide evidence that gp79, a currently unknown protein of ϕCh1, acts as a repressor protein of the viral life cycle, affecting the transition from the lysogenic to the lytic state of the virus. Thus, repressor genes in other haloviruses could be identified by sequence homologies to gp79 in the future. Moreover, we describe the use of an inducible promoter of N. magadii Our work provides valuable tools for the identification of other unknown viral genes by our approach as well as for functional studies of proteins by inducible expression.

Disk patch resonators for cavity quantum electrodynamics at the terahertz frequency.

We designed disk patch resonators to meet the requirements for enhanced coupling of optical cavities to intersubband transitions in heterostructures in the terahertz frequency regime. We applied modifications to the standard patch resonator in the form of a chain of holes and slits to control the resonator eigenmodes featuring quality factors ωFWHM/ω0 as high as 40. Due to the broken rotational symmetry of the resonators the individual eigenmodes can be accessed selectively depending on the incidence and the polarization of the THz wave. The demonstrated post-process blue-shifting of the resonance frequency up to 50% is a key tuning knob for an optimization of light-matter interaction in a quantum system.

Identification of the Main Regulator Responsible for Synthesis of the Typical Yellow Pigment Produced by Trichoderma reesei.

The industrially used ascomycete Trichoderma reesei secretes a typical yellow pigment during cultivation, while other Trichoderma species do not. A comparative genomic analysis suggested that a putative secondary metabolism cluster, containing two polyketide-synthase encoding genes, is responsible for the yellow pigment synthesis. This cluster is conserved in a set of rather distantly related fungi, including Acremonium chrysogenum and Penicillium chrysogenum In an attempt to silence the cluster in T. reesei, two genes of the cluster encoding transcription factors were individually deleted. For a complete genetic proof-of-function, the genes were reinserted into the genomes of the respective deletion strains. The deletion of the first transcription factor (termed yellow pigment regulator 1 [Ypr1]) resulted in the full abolishment of the yellow pigment formation and the expression of most genes of this cluster. A comparative high-pressure liquid chromatography (HPLC) analysis of supernatants of the ypr1 deletion and its parent strain suggested the presence of several yellow compounds in T. reesei that are all derived from the same cluster. A subsequent gas chromatography/mass spectrometry analysis strongly indicated the presence of sorbicillin in the major HPLC peak. The presence of the second transcription factor, termed yellow pigment regulator 2 (Ypr2), reduces the yellow pigment formation and the expression of most cluster genes, including the gene encoding the activator Ypr1. IMPORTANCE: Trichoderma reesei is used for industry-scale production of carbohydrate-active enzymes. During growth, it secretes a typical yellow pigment. This is not favorable for industrial enzyme production because it makes the downstream process more complicated and thus increases operating costs. In this study, we demonstrate which regulators influence the synthesis of the yellow pigment. Based on these data, we also provide indication as to which genes are under the control of these regulators and are finally responsible for the biosynthesis of the yellow pigment. These genes are organized in a cluster that is also found in other industrially relevant fungi, such as the two antibiotic producers Penicillium chrysogenum and Acremonium chrysogenum The targeted manipulation of a secondary metabolism cluster is an important option for any biotechnologically applied microorganism.

The Relation Between Promoter Chromatin Status, Xyr1 and Cellulase Ex-pression in Trichoderma reesei.

The ascomycete Trichoderma reesei is used for the production of plant cell wall-degrading enzymes in industrial scale. The interplay of the transactivator Xyr1 and the repressor Cre1 mainly regulates the expression of these enzymes. During induc-ing conditions, such as in the presence of sophorose, the transcription of the two major cellulase-encoding genes, cbh1 and cbh2, is activated as well as the expression of xyr1. In the presence of D-glucose carbon catabolite repression mediated by Cre1 takes place and the expression of Xyr1 and the plant cell wall-degrading enzymes is down-regulated. In this study we compare the chromatin status of xyr1, cbh1, and cbh2 promoters in the wild-type strain and the Cre1-deficient strain Rut-C30. Chromatin rearrangement occurs in the xyr1 promoter during induction on sophorose. Chromatin opening and protein-DNA interactions in the xyr1 promoter were detected especially in a region located 0.9 kb upstream the translation start co-don, which bears several putative Cre1-binding sites and a CCAAT-box. Moreover, the xyr1 promoter is overall more acces-sible in a cre1-truncated background, no matter which carbon source is present. This makes the xyr1 regulatory sequence a good target for promoter engineering aiming at the enhancement of cellulase production.

Novel Strategies for Genomic Manipulation of Trichoderma reesei with the Purpose of Strain Engineering.

The state-of-the-art procedure for gene insertions into Trichoderma reesei is a cotransformation of two plasmids, one bearing the gene of interest and the other a marker gene. This procedure yields up to 80% transformation efficiency, but both the number of integrated copies and the loci of insertion are unpredictable. This can lead to tremendous pleiotropic effects. This study describes the development of a novel transformation system for site-directed gene insertion based on auxotrophic markers. For this purpose, we tested the applicability of the genes asl1 (encoding an enzyme of the l-arginine biosynthesis pathway), the hah1 (encoding an enzyme of the l-lysine biosynthesis pathway), and the pyr4 (encoding an enzyme of the uridine biosynthesis pathway). The developed transformation system yields strains with an additional gene at a defined locus that are prototrophic and ostensibly isogenic compared to their parental strain. A positive transformation rate of 100% was achieved due to the developed split-marker system. Additionally, a double-auxotrophic strain that allows multiple genomic manipulations was constructed, which facilitates metabolic engineering purposes in T. reesei. By employing goxA of Aspergillus niger as a reporter system, the influence on the expression of an inserted gene caused by the orientation of the insertion and the transformation strategy used could be demonstrated. Both are important aspects to be considered during strain engineering.

Arrays of open, independently tunable microcavities.

Optical cavities are of central importance in numerous areas of physics, including precision measurement, cavity optomechanics and cavity quantum electrodynamics. The miniaturisation and scaling to large numbers of sites is of interest for many of these applications, in particular for quantum computation and simulation. Here we present the first scaled microcavity system which enables the creation of large numbers of highly uniform, tunable light-matter interfaces using ions, neutral atoms or solid-state qubits. The microcavities are created by means of silicon micro-fabrication, are coupled directly to optical fibres and can be independently tuned to the chosen frequency, paving the way for arbitrarily large networks of optical microcavities.

Use of Auxotrophic Markers for Targeted Gene Insertions in Trichoderma reesei.

In this chapter, we describe a routinely used strategy for targeted gene insertions in Trichoderma reesei using auxotrophic markers. Generally, targeted gene integrations are advantageous over random, ectopic integration, because the copy number and locus of integration are controlled, abolishing the risk of pleiotropic effects. The use of auxotrophic markers allows a direct, cheap, and easy method for selection. The first step is the construction of recipient strains in a NHEJ-deficient strain. We routinely use deletion strains of pyr4, encoding for the orotidine 5'-phosphate decarboxylase (EC 4.1.1.23) and/or asl1, encoding for the argininosuccinate lyase (EC 4.3.2.1). In the second step, the gene of interest is inserted together with the marker gene. Here we describe the necessary strategy for the construction of the recipient strains and insertion constructs, a PEG-mediated transformation protocol, and a protocol for genetic confirmation of the gene insertion.

Expanding the toolbox: another auxotrophic marker for targeted gene integrations in Trichoderma reesei.

BACKGROUND: The filamentous ascomycete Trichoderma reesei is used for the industrial production of cellulases and holds the promise for heterologous gene expression due to its outstandingly high protein secretion rates and its long-term application in industry and science. A prerequisite for successful heterologous gene expression is the ability to insert a corresponding expression cassette at suitable loci in the genome of T. reesei. RESULTS: In this study, we test and demonstrate the applicability of the his1 gene [encoding for the ATP phosphoribosyltransferase (EC 2.4.2.17), part of the histidine biosynthesis pathway] and locus for targeted gene insertion. Deletion of the his1 promoter and a part of the coding region leads to histidine auxotrophy. Reestablishment of the his1 locus restores prototrophy. We designed a matching plasmid that allows integration of an expression cassette at the his1 locus. This is demonstrated by the usage of the reporter EYFP (enhanced yellow fluorescence protein). Further, we describe a minimal effort and seamless marker recycling method. Finally, we test the influence of the integration site on the gene expression by comparing three strains bearing the same EYFP expression construct at different loci. CONCLUSION: With the establishment of his1 as integration locus and auxotrophic marker, we could expand the toolbox for strain design in T. reesei. This facilitates future strain constructions with the aim of heterologous gene expression.

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.

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.

Application of the human estrogen receptor within a synthetic transcription factor in Trichoderma reesei.

BACKGROUND: Synthetic gene expression systems offer a possibility for controllable and targeted induction of the expression of genes of interest, which is a fundamental technique necessary for basic research and industrial applications. The human estrogen receptor α contains a ligand binding domain that enforces dimerization and nuclear import upon binding of the inducer 17ß-estradiol. In this study, we tested the potential of this ligand binding domain to be used in filamentous fungi as an auto-regulatory domain in a synthetic transcription factor. RESULTS: We constructed the synthetic transcription factor SynX by fusing the DNA-binding domain of Xyr1 (Xylanase Regulator 1), the transactivation domain of Ypr1 (Yellow Pigment Regulator 1), and the ligand binding domain of the human estrogen receptor α. SynX is able to strongly induce the gene expression of xylanases and an aldose reductase by addition of 17ß-estradiol, but SynX does not induce gene expression of cellulases. Importantly, the induction of xylanase activities is mostly carbon source independent and can be fine-tuned by controlling the concentration of 17ß-estradiol. CONCLUSION: The ability of SynX to induce gene expression of xylanase encoding genes by addition of 17ß-estradiol demonstrates that the ligand binding domain of the human estrogen receptor α works in filamentous fungi, and that it can be combined with a transactivation domain other than the commonly used transactivation domain of herpes simplex virion protein VP16.

Fusion transcription factors for strong, constitutive expression of cellulases and xylanases in Trichoderma reesei.

BACKGROUND: The filamentous ascomycete T. reesei is industrially used to produce cellulases and xylanases. Cost-effective production of cellulases is a bottleneck for biofuel production. Previously, different strain and process optimizations were deployed to enhance enzyme production rates. One approach is the overexpression of the main activator Xyr1 and a second is the construction of synthetic transcription factors. Notably, these genetic manipulations were introduced into strains bearing the wild-type xyr1 gene and locus. RESULTS: Here, we constructed a Xyr1-deficient strain expressing a non-functional truncated version of Xyr1. This strain was successfully used as platform strain for overexpression of Xyr1, which enhanced the cellulase and xylanase production rates under inducing conditions, with the exception of lactose-there the cellulase production was severely reduced. Further, we introduced fusion transcription factors consisting of the DNA-binding domain of Xyr1 and the transactivation domain of either Ypr1 or Ypr2 (regulators of the sorbicillinoid biosynthesis gene cluster). The fusion of Xyr1 and Ypr2 yielded a moderately transactivating transcription factor, whereas the fusion of Xyr1 and Ypr1 yielded a highly transactivating transcription factor that induced xylanases and cellulases nearly carbon source independently. Especially, high production levels of xylanases were achieved on glycerol. CONCLUSION: During this study, we constructed a Xyr1-deficient strain that can be fully reconstituted, which makes it an ideal platform strain for Xyr1-related studies. The mere overexpression of Xyr1 turned out not to be a successful strategy for overall enhancement of the enzyme production rates. We gained new insights into the regulatory properties of transcription factors by constructing respective fusion proteins. The Xyr1-Ypr1-fusion transcription factor could induce xylanase production rates on glycerol to outstanding extents, and hence could be deployed in the future to utilize crude glycerol, the main co-product of the biodiesel production process.

In Vivo Study of the Sorbicillinoid Gene Cluster in Trichoderma reesei.

Sorbicillinoids are a diverse group of yellow secondary metabolites that are produced by a range of not closely related ascomycetes, including Penicillium chrysogenum, Acremonium chrysogenum, and Trichoderma reesei. They share a similarity to the name-giving compound sorbicillin, a hexaketide. Previously, a conserved gene cluster containing two polyketide synthases has been identified as the source of sorbicillin, and a model for the biosynthesis of sorbicillin in P. chrysogenum has been proposed. In this study, we deleted the major genes of interest of the cluster in T. reesei, namely sor1, sor3, and sor4. Sor1 is the homolog of P. chrysogenum SorA, which is the first polyketide synthase of the proposed biosynthesis pathway. Sor3 is a flavin adenine dinucleotide (FAD)-dependent monooxygenase, and its homolog in P. chrysogenum, SorC, was shown to oxidize sorbicillin and 2',3'-dihydrosorbicillin to sorbicillinol and 2',3'-dihydrosorbicillinol, respectively, in vitro. Sor4 is an FAD/flavin mononucleotide-containing dehydrogenase with an unknown function. We measured the amounts of synthesized sorbicillinoids throughout growth and could verify the roles of Sor1 and Sor3 in vivo in T. reesei. In the absence of Sor4, two compounds annotated to dihydrosorbicillinol accumulate in the supernatant and only small amounts of sorbicillinol are synthesized. Therefore, we suggest extending the current biosynthesis model about Sor4 reducing 2',3'-dihydrosorbicillin and 2',3'-dihydrosorbicillinol to sorbicillinol and sorbicillinol, respectively. Sorbicillinol turned out to be the main chemical building block for most sorbicillinoids, including oxosorbicillinol, bisorbicillinol, and bisvertinolon. Further, we detected the sorbicillinol-dependent synthesis of 5-hydroxyvertinolide at early time points, which contradicts previous models for biosynthesis of 5-hydroxyvertinolide. Finally, we investigated whether sorbicillinoids from T. reesei have a growth limiting effect on the fungus itself or on plant pathogenic fungi or on pathogenic bacteria.