Whole-genome metabolic model of <Emphasis Type="Italic">Trichoderma reesei</Emphasis> built by comparative reconstruction

Background

Trichoderma reesei is one of the main sources of biomass-hydrolyzing enzymes for the biotechnology industry. There is a need for improving its enzyme production efficiency. The use of metabolic modeling for the simulation and prediction of this organism’s metabolism is potentially a valuable tool for improving its capabilities. An accurate metabolic model is needed to perform metabolic modeling analysis.

Results

A whole-genome metabolic model of T. reesei has been reconstructed together with metabolic models of 55 related species using the metabolic model reconstruction algorithm CoReCo. The previously published CoReCo method has been improved to obtain better quality models. The main improvements are the creation of a unified database of reactions and compounds and the use of reaction directions as constraints in the gap-filling step of the algorithm. In addition, the biomass composition of T. reesei has been measured experimentally to build and include a specific biomass equation in the model.

Conclusions

The improvements presented in this work on the CoReCo pipeline for metabolic model reconstruction resulted in higher-quality metabolic models compared with previous versions. A metabolic model of T. reesei has been created and is publicly available in the BIOMODELS database. The model contains a biomass equation, reaction boundaries and uptake/export reactions which make it ready for simulation. To validate the model, we dem1onstrate that the model is able to predict biomass production accurately and no stoichiometrically infeasible yields are detected. The new T. reesei model is ready to be used for simulations of protein production processes.

     

Arsenal of plant cell wall degrading enzymes reflects host preference among plant pathogenic fungi

Background  

The discovery and development of novel plant cell wall degrading enzymes is a key step towards more efficient depolymerization of polysaccharides to fermentable sugars for the production of liquid transportation biofuels and other bioproducts. The industrial fungus Trichoderma reesei is known to be highly cellulolytic and is a major industrial microbial source for commercial cellulases, xylanases and other cell wall degrading enzymes. However, enzyme-prospecting research continues to identify opportunities to enhance the activity of T. reesei enzyme preparations by supplementing with enzymatic diversity from other microbes. The goal of this study was to evaluate the enzymatic potential of a broad range of plant pathogenic and non-pathogenic fungi for their ability to degrade plant biomass and isolated polysaccharides.     

Cocktail δ-integration: a novel method to construct cellulolytic enzyme expression ratio-optimized yeast strains

Background  

The filamentous fungus T. reesei effectively degrades cellulose and is known to produce various cellulolytic enzymes such as β-glucosidase, endoglucanase, and cellobiohydrolase. The expression levels of each cellulase are controlled simultaneously, and their ratios and synergetic effects are important for effective cellulose degradation. However, in recombinant Saccharomyces cerevisiae, it is difficult to simultaneously control many different enzymes. To construct engineered yeast with efficient cellulose degradation, we developed a simple method to optimize cellulase expression levels, named cocktail δ-integration.     

The genome-scale metabolic network analysis <Emphasis Type="Italic">of Zymomonas mobilis</Emphasis> ZM4 explains physiological features and suggests ethanol and succinic acid production strategies

Background  

Zymomonas mobilis ZM4 is a Gram-negative bacterium that can efficiently produce ethanol from various carbon substrates, including glucose, fructose, and sucrose, via the Entner-Doudoroff pathway. However, systems metabolic engineering is required to further enhance its metabolic performance for industrial application. As an important step towards this goal, the genome-scale metabolic model of Z. mobilis is required to systematically analyze in silico the metabolic characteristics of this bacterium under a wide range of genotypic and environmental conditions.     

Using a logical model to predict the growth of yeast

Background  

A logical model of the known metabolic processes in S. cerevisiae was constructed from iFF708, an existing Flux Balance Analysis (FBA) model, and augmented with information from the KEGG online pathway database. The use of predicate logic as the knowledge representation for modelling enables an explicit representation of the structure of the metabolic network, and enables logical inference techniques to be used for model identification/improvement.     

Sulphur metabolism and cellulase gene expression are connected processes in the filamentous fungus <Emphasis Type="Italic">Hypocrea jecorina</Emphasis> (anamorph <Emphasis Type="Italic">Trichoderma reesei</Emphasis>)

Background  

Sulphur compounds like cysteine, methionine and S-adenosylmethionine are essential for the viability of most cells. Thus many organisms have developed a complex regulatory circuit that governs the expression of enzymes involved in sulphur assimilation and metabolism. In the filamentous fungus Hypocrea jecorina (anamorph Trichoderma reesei) little is known about the participants in this circuit.     

Evolution and Ecophysiology of the Industrial Producer Hypocrea jecorina (Anamorph Trichoderma reesei) and a New Sympatric Agamospecies Related to It

Background

Trichoderma reesei, a mitosporic green mould, was recognized during the WW II based on a single isolate from the Solomon Islands and since then used in industry for production of cellulases. It is believed to be an anamorph (asexual stage) of the common pantropical ascomycete Hypocrea jecorina.

Methodology/Principal Findings

We combined molecular evolutionary analysis and multiple methods of phenotype profiling in order to reveal the genetic relationship of T. reesei to H. jecorina. The resulting data show that the isolates which were previously identified as H. jecorina by means of morphophysiology and ITS1 and 2 (rRNA gene cluster) barcode in fact comprise several species: i) H. jecorina/T. reesei sensu stricto which contains most of the teleomorphs (sexual stages) found on dead wood and the wild-type strain of T. reesei QM 6a; ii) T. parareesei nom. prov., which contains all strains isolated as anamorphs from soil; iii) and two other hypothetical new species for which only one or two isolates are available. In silico tests for recombination and in vitro mating experiments revealed a history of sexual reproduction for H. jecorina and confirmed clonality for T. parareesei nom. prov. Isolates of both species were consistently found worldwide in pantropical climatic zone. Ecophysiological comparison of H. jecorina and T. parareesei nom. prov. revealed striking differences in carbon source utilization, conidiation intensity, photosensitivity and mycoparasitism, thus suggesting adaptation to different ecological niches with the high opportunistic potential for T. parareesei nom. prov.

Conclusions

Our data prove that T. reesei belongs to a holomorph H. jecorina and displays a history of worldwide gene flow. We also show that its nearest genetic neighbour - T. parareesei nom. prov., is a cryptic phylogenetic agamospecies which inhabits the same biogeographic zone. These two species thus provide a so far rare example of sympatric speciation within saprotrophic fungi, with divergent ecophysiological adaptations and reproductive strategies.     

Heterologous expression of an endo-β-1,4-glucanase gene from the anaerobic fungus <Emphasis Type="Italic">Orpinomyces</Emphasis> PC-2 in <Emphasis Type="Italic">Trichoderma reesei</Emphasis>

The codon modified neutral endo-β-1,4-glucanase gene celEn, originating from the anaerobic fungus Orpinomyces sp. strain PC-2, was inserted between the strong promoter Pcel7A and the terminator Tcel7A from Trichoderma reesei. The resulting expression cassette was ligated to the pCAMBIA1300 Agrobacterium binary vector to construct pCB-hE that also contains a hygromycin B resistance marker. pCB-hE was introduced into T. reesei ZU-02 through an Agrobacterium tumefaciens–mediated transformation procedure that has been modified with an improved transformation efficiency of 12,500 transformants per 10⁷ conidia. Stable integration of the celEn gene into the chromosomal DNA of T. reesei ZU-02 was confirmed by PCR. After 48 h fermentation in shaking flasks, the endo-β-1,4-glucanase activities increased to 55–70 IU ml⁻¹ in transgenic strains, which were about 6–7 times higher than that of the original ZU-02 strain (9.5 IU ml⁻¹). When the avicel was added in fermentation medium, the endo-β-1,4-glucanase activity in the transgenic strains could be further increased to 193.6 IU ml⁻¹ after 84 h fermentation. Transgenic T. reesei strains with high neutral endo-β-1,4-glucanase activity will be particularly suitable for certain applications in textile industry. The improved procedures for overproduction and secretion of heterologous proteins in transgenic T. reesei can also be used to generate similar recombinant proteins for research or industrial purposes.     

Light-dependent roles of the G-protein α subunit GNA1 of Hypocrea jecorina (anamorph Trichoderma reesei)

Background  

The filamentous ascomycete Hypocrea jecorina (anamorph Trichoderma reesei) is primarily known for its efficient enzymatic machinery that it utilizes to decompose cellulosic substrates. Nevertheless, the nature and transmission of the signals initiating and modulating this machinery are largely unknown. Heterotrimeric G-protein signaling represents one of the best studied signal transduction pathways in fungi.     

Enzymatic hydrolyzing performance of Acremonium cellulolyticus and Trichoderma reesei against three lignocellulosic materials

Background  

Bioethanol isolated from lignocellulosic biomass represents one of the most promising renewable and carbon neutral alternative liquid fuel sources. Enzymatic saccharification using cellulase has proven to be a useful method in the production of bioethanol. The filamentous fungi Acremonium cellulolyticus and Trichoderma reesei are known to be potential cellulase producers. In this study, we aimed to reveal the advantages and disadvantages of the cellulase enzymes derived from these fungi.     

A versatile toolkit for high throughput functional genomics with Trichoderma reesei

Background  

The ascomycete fungus, Trichoderma reesei (anamorph of Hypocrea jecorina), represents a biotechnological workhorse and is currently one of the most proficient cellulase producers. While strain improvement was traditionally accomplished by random mutagenesis, a detailed understanding of cellulase regulation can only be gained using recombinant technologies.     

Lactate production yield from engineered yeasts is dependent from the host background, the lactate dehydrogenase source and the lactate export

Background  

Metabolic pathway manipulation for improving the properties and the productivity of microorganisms is becoming a well established concept. For the production of important metabolites, but also for a better understanding of the fundamentals of cell biology, detailed studies are required. In this work we analysed the lactate production from metabolic engineered Saccharomyces cerevisiae cells expressing a heterologous lactate dehydrogenase (LDH) gene. The LDH gene expression in a budding yeast cell introduces a novel and alternative pathway for the NAD⁺ regeneration, allowing a direct reduction of the intracellular pyruvate to lactate, leading to a simultaneous accumulation of lactate and ethanol.     

Comparative secretome analyses of two <Emphasis Type="Italic">Trichoderma reesei</Emphasis> RUT-C30 and CL847 hypersecretory strains

Background  

Due to its capacity to produce large amounts of cellulases, Trichoderma reesei is increasingly been researched in various fields of white biotechnology, especially in biofuel production from lignocellulosic biomass. The commercial enzyme mixtures produced at industrial scales are not well characterized, and their proteinaceous components are poorly identified and quantified. The development of proteomic methods has made it possible to comprehensively overview the enzymes involved in lignocellulosic biomass degradation which are secreted under various environmental conditions.     

Transglycosylation products of cellulase system ofTrichoderma reesei

Summary From cellulose and cellobiose the formation of sophorose, laminaribiose, and gentiobiose was catalyzed byTrichoderma reesei culture filtrate containing exo- and endoglucanase and -glucosidase activity and from cellobiose by a broken cell suspension fromT.reesei with -glucosidase activity. The results indicate that -glucosidase is the component responsible for transglycosylation reaction catalyzed byT.reesei cellulase enzyme complex.     

Addendum to Issue 1 - ENZITEC 2012Simultaneous biosynthesis of biomass-degrading enzymes using co-cultivation of Aspergillus niger and Trichoderma reesei

Abstract

The biotransformation of lignocellulosic materials into biofuels and chemicals requires the simultaneous action of multiple enzymes. Since the cost of producing an efficient enzyme system maybe high, mixed cultures of microorganisms maybe an alternative to increase enzymatic production and consequently reduce costs. This study investigated the effects of different inoculum ratios and inoculation delays on the biosynthesis of cellulases and xylanases during co-cultivation of Aspergillus niger and Trichoderma reesei under solid-state fermentation (SSF). While the monoculture of T. reesei was more efficient for CMCase production than the co-cultivation of A. niger and T. reesei, a significant increase in β-glucosidase and xylanase production was achieved by co-cultivation of both species. The maximum CMCase activity of 153.91 IU/g was obtained with T. reesei after 48 h of cultivation, while the highest β-glucosidase activity of 119.71 IU/g (after 120 h) was obtained by co-cultivation of A. niger and T. reesei with a 3:1 inoculum ratio (A. niger: T. reesei). The greatest xylanase activity observed was 589.39 IU/g after 72 h of mixed culturing of A. niger and T. Reesei with a 1:1 inoculum ratio. This is the first study where the effects of inoculum ratio and inoculation delay in mixed culture of T. reesei and A. niger under SSF have been systematically assessed, and it indicates co-cultivation as a feasible alternative to increase enzymatic production.     

The Golgi α-1,6 mannosyltransferase KlOch1p of Kluyveromyces lactisis required for Ca2+/calmodulin-based signaling and for proper mitochondrial functionality

Background  

ProteinN-glycosylation is a relevant metabolic pathway in eukaryotes and plays key roles in cell processes. In yeasts, outer chain branching is initiated in the Golgi apparatus by the alpha-1,6-mannosyltransferase Och1p.     

A versatile <Emphasis Type="Italic">Trichoderma reesei</Emphasis> expression system for the production of heterologous proteins

Objectives

To develop a versatile Trichoderma reesei (teleomorph Hypocrea jecorina) expression system for the high-purity production of heterologous proteins.

Results

The versatile T. reesei expression system is based on xyn1 and xyn2 promoters, A824V transition in XYRI, and a bicomponent carbon source strategy. Red fluorescent protein gene rfp and alkaline endoglucanase EGV gene egv3 from Humicola insolens were used as reporter genes to test our versatile expression system

Conclusions

The versatile T. reesei expression system can be applied to produce heterologous proteins with high purity and high yield.

     

Mutagenesis of Trichoderma reesei endoglucanase I: impact of expression host on activity and stability at elevated temperatures

Background

Trichoderma reesei is a key cellulase source for economically saccharifying cellulosic biomass for the production of biofuels. Lignocellulose hydrolysis at temperatures above the optimum temperature of T. reesei cellulases (~50°C) could provide many significant advantages, including reduced viscosity at high-solids loadings, lower risk of microbial contamination during saccharification, greater compatibility with high-temperature biomass pretreatment, and faster rates of hydrolysis. These potential advantages motivate efforts to engineer T. reesei cellulases that can hydrolyze lignocellulose at temperatures ranging from 60–70°C.

Results

A B-factor guided approach for improving thermostability was used to engineer variants of endoglucanase I (Cel7B) from T. reesei (TrEGI) that are able to hydrolyze cellulosic substrates more rapidly than the recombinant wild-type TrEGI at temperatures ranging from 50–70°C. When expressed in T. reesei, TrEGI variant G230A/D113S/D115T (G230A/D113S/D115T Tr_TrEGI) had a higher apparent melting temperature (3°C increase in T_m) and improved half-life at 60°C (t_1/2 = 161 hr) than the recombinant (T. reesei host) wild-type TrEGI (t_1/2 = 74 hr at 60°C, Tr_TrEGI). Furthermore, G230A/D113S/D115T Tr_TrEGI showed 2-fold improved activity compared to Tr_TrEGI at 65°C on solid cellulosic substrates, and was as efficient in hydrolyzing cellulose at 60°C as Tr_TrEGI was at 50°C. The activities and stabilities of the recombinant TrEGI enzymes followed similar trends but differed significantly in magnitude depending on the expression host (Escherichia coli cell-free, Saccharomyces cerevisiae, Neurospora crassa, or T. reesei). Compared to N.crassa-expressed TrEGI, S. cerevisiae-expressed TrEGI showed inferior activity and stability, which was attributed to the lack of cyclization of the N-terminal glutamine in Sc_TrEGI and not to differences in glycosylation. N-terminal pyroglutamate formation in TrEGI expressed in S. cerevisiae was found to be essential in elevating its activity and stability to levels similar to the T. reesei or N. crassa-expressed enzyme, highlighting the importance of this ubiquitous modification in GH7 enzymes.

Conclusion

Structure-guided evolution of T. reesei EGI was used to engineer enzymes with increased thermal stability and activity on solid cellulosic substrates. Production of TrEGI enzymes in four hosts highlighted the impact of the expression host and the role of N-terminal pyroglutamate formation on the activity and stability of TrEGI enzymes.

Electronic supplementary material

The online version of this article (doi:10.1186/s12896-015-0118-z) contains supplementary material, which is available to authorized users.     

Fermentation of mixed glucose-xylose substrates by engineered strains of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>: role of the coenzyme specificity of xylose reductase,and effect of glucose on xylose utilization

Background  

In spite of the substantial metabolic engineering effort previously devoted to the development of Saccharomyces cerevisiae strains capable of fermenting both the hexose and pentose sugars present in lignocellulose hydrolysates, the productivity of reported strains for conversion of the naturally most abundant pentose, xylose, is still a major issue of process efficiency. Protein engineering for targeted alteration of the nicotinamide cofactor specificity of enzymes catalyzing the first steps in the metabolic pathway for xylose was a successful approach of reducing xylitol by-product formation and improving ethanol yield from xylose. The previously reported yeast strain BP10001, which expresses heterologous xylose reductase from Candida tenuis in mutated (NADH-preferring) form, stands for a series of other yeast strains designed with similar rational. Using 20 g/L xylose as sole source of carbon, BP10001 displayed a low specific uptake rate q _xylose (g xylose/g dry cell weight/h) of 0.08. The study presented herein was performed with the aim of analysing (external) factors that limit q _xylose of BP10001 under xylose-only and mixed glucose-xylose substrate conditions. We also carried out a comprehensive investigation on the currently unclear role of coenzyme utilization, NADPH compared to NADH, for xylose reduction during co-fermentation of glucose and xylose.