Whole-genome metabolic model of Trichoderma reesei built by comparative reconstruction

Castillo, Sandra; Barth, Dorothee; Arvas, Mikko; Pakula, Tiina; Pitkänen, Esa; Blomberg, Peter; Seppänen‐Laakso, Tuulikki; Nygrén, Heli; Sivasiddarthan, Dhinakaran; Penttilä, Merja; Oja, Merja

doi:10.1186/s13068-016-0665-0

Cited by 24 publications

(24 citation statements)

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“…In general, an inverse correlation between citric acid/protein production and cell growth has been reported for A. niger [48, 49]. In agreement, a high specific protein production rate is also achieved at relatively low growth rates in the protein cell factory Trichoderma reesei [50]. Therefore, our data are in agreement with these reports and suggest that the efficient development of filamentous fungi as microbial cell factories requires integrative genetic engineering approaches which couple different growth-related and product related processes.…”

Section: Discussionmentioning

confidence: 75%

Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture

Cairns

Feurstein

Zheng

et al. 2019

Fungal Biol Biotechnol

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BackgroundFilamentous fungal cell factories are used to produce numerous proteins, enzymes, and organic acids. Protein secretion and filamentous growth are tightly coupled at the hyphal tip. Additionally, both these processes require ATP and amino acid precursors derived from the citric acid cycle. Despite this interconnection of organic acid production and protein secretion/filamentous growth, few studies in fungi have identified genes which may concomitantly impact all three processes.ResultsWe applied a novel screen of a global co-expression network in the cell factory Aspergillus niger to identify candidate genes which may concomitantly impact macromorphology, and protein/organic acid fermentation. This identified genes predicted to encode the Golgi localized ArfA GTPase activating protein (GAP, AgeB), and ArfA guanine nucleotide exchange factors (GEFs SecG and GeaB) to be co-expressed with citric acid cycle genes. Consequently, we used CRISPR-based genome editing to place the titratable Tet-on expression system upstream of ageB, secG, and geaB in A. niger. Functional analysis revealed that ageB and geaB are essential whereas secG was dispensable for early filamentous growth. Next, gene expression was titrated during submerged cultivations under conditions for either protein or organic acid production. ArfA regulators played varied and culture-dependent roles on pellet formation. Notably, ageB or geaB expression levels had major impacts on protein secretion, whereas secG was dispensable. In contrast, reduced expression of each predicted ArfA regulator resulted in an absence of citric acid in growth media. Finally, titrated expression of either GEFs resulted in an increase in oxaloacetic acid concentrations in supernatants.ConclusionOur data suggest that the Golgi may play an underappreciated role in modulating organic acid titres during industrial applications, and that this is SecG, GeaB and AgeB dependent in A. niger. These data may lead to novel avenues for strain optimization in filamentous fungi for improved protein and organic acid titres.

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Section: Discussionmentioning

confidence: 75%

Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture

Cairns

Feurstein

Zheng

et al. 2019

Fungal Biol Biotechnol

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“…This observation is generally consistent with previous reports for A. niger and other filamentous fungal cell factories. For example, a high specific protein production rate has been shown to correlate with relatively low growth rates in Trichoderma reesei [ 49 ], due to a reduced proteome allocated for central metabolism [ 12 ]. Also, a recent multi-omics analysis from our group that integrated transcriptomics, metabolomics and GSMM simulations proposed that an increased GlaA production is probably achieved through reduced growth, which likely is regulated at several metabolic mechanisms: (i) an increased carbon catabolism that generates more amino acid precursors for protein production, (ii) a reduced fatty acid and ribosome biogenesis and, thus, reduced growth, and (iii) an increased flux through the glyoxylate bypass to reduce NADH formation from the citric acid cycle and to maintain the cellular redox balance [ 50 ].…”

Section: Discussionmentioning

confidence: 99%

Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger

et al. 2020

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Background Nicotinamide adenine dinucleotide phosphate (NADPH) is an important cofactor ensuring intracellular redox balance, anabolism and cell growth in all living systems. Our recent multi-omics analyses of glucoamylase (GlaA) biosynthesis in the filamentous fungal cell factory Aspergillus niger indicated that low availability of NADPH might be a limiting factor for GlaA overproduction. Results We thus employed the Design-Build-Test-Learn cycle for metabolic engineering to identify and prioritize effective cofactor engineering strategies for GlaA overproduction. Based on available metabolomics and 13C metabolic flux analysis data, we individually overexpressed seven predicted genes encoding NADPH generation enzymes under the control of the Tet-on gene switch in two A. niger recipient strains, one carrying a single and one carrying seven glaA gene copies, respectively, to test their individual effects on GlaA and total protein overproduction. Both strains were selected to understand if a strong pull towards glaA biosynthesis (seven gene copies) mandates a higher NADPH supply compared to the native condition (one gene copy). Detailed analysis of all 14 strains cultivated in shake flask cultures uncovered that overexpression of the gsdA gene (glucose 6-phosphate dehydrogenase), gndA gene (6-phosphogluconate dehydrogenase) and maeA gene (NADP-dependent malic enzyme) supported GlaA production on a subtle (10%) but significant level in the background strain carrying seven glaA gene copies. We thus performed maltose-limited chemostat cultures combining metabolome analysis for these three isolates to characterize metabolic-level fluctuations caused by cofactor engineering. In these cultures, overexpression of either the gndA or maeA gene increased the intracellular NADPH pool by 45% and 66%, and the yield of GlaA by 65% and 30%, respectively. In contrast, overexpression of the gsdA gene had a negative effect on both total protein and glucoamylase production. Conclusions This data suggests for the first time that increased NADPH availability can indeed underpin protein and especially GlaA production in strains where a strong pull towards GlaA biosynthesis exists. This data also indicates that the highest impact on GlaA production can be engineered on a genetic level by increasing the flux through the pentose phosphate pathway (gndA gene) followed by engineering the flux through the reverse TCA cycle (maeA gene). We thus propose that NADPH cofactor engineering is indeed a valid strategy for metabolic engineering of A. niger to improve GlaA production, a strategy which is certainly also applicable to the rational design of other microbial cell factories.

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“…To this end, Castillo et al . () applied the CoReCo (comparative metabolic reconstruction framework) pipeline (Pitkänen et al ., ) for the construction of a high‐quality metabolic model of T. reesei (BIOMODELS database). The model contains a biomass equation, reaction boundaries and uptake/export reactions, which make it ready for the simulation of protein production processes.…”

Section: Engineering Cellulase Gene Expressionmentioning

confidence: 99%

Genetic engineering of Trichoderma reesei cellulases and their production

Druzhinina

Kubicek

2017

Microbial Biotechnology

178

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SummaryLignocellulosic biomass, which mainly consists of cellulose, hemicellulose and lignin, is the most abundant renewable source for production of biofuel and biorefinery products. The industrial use of plant biomass involves mechanical milling or chipping, followed by chemical or physicochemical pretreatment steps to make the material more susceptible to enzymatic hydrolysis. Thereby the cost of enzyme production still presents the major bottleneck, mostly because some of the produced enzymes have low catalytic activity under industrial conditions and/or because the rate of hydrolysis of some enzymes in the secreted enzyme mixture is limiting. Almost all of the lignocellulolytic enzyme cocktails needed for the hydrolysis step are produced by fermentation of the ascomycete Trichoderma reesei (Hypocreales). For this reason, the structure and mechanism of the enzymes involved, the regulation of their expression and the pathways of their formation and secretion have been investigated in T. reesei in considerable details. Several of the findings thereby obtained have been used to improve the formation of the T. reesei cellulases and their properties. In this article, we will review the achievements that have already been made and also show promising fields for further progress.

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Whole-genome metabolic model of Trichoderma reesei built by comparative reconstruction

Cited by 24 publications

References 54 publications

Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture

Functional exploration of co-expression networks identifies a nexus for modulating protein and citric acid titres in Aspergillus niger submerged culture

Engineering cofactor metabolism for improved protein and glucoamylase production in Aspergillus niger

Genetic engineering of Trichoderma reesei cellulases and their production

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