Genetic engineering and raising temperature enhance recombinant protein production with the cdna1 promoter in Trichoderma reesei
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ABSTRACT:
SUBMITTER: Jiang S
PROVIDER: S-EPMC10991654 | biostudies-literature | 2022 Oct
REPOSITORIES: biostudies-literature
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Project description:Lignocellulosic 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.
| S-EPMC5658622 | biostudies-literature
Project description:BackgroundTrichoderma reesei is the preferred organism for producing industrial cellulases. However, a more efficient heterologous expression system for enzymes from different organism is needed to further improve its cellulase mixture. The strong cbh1 promoter of T. reesei is frequently used in heterologous expression, however, the carbon catabolite repressor CREI may reduce its strength by binding to the cbh1 promoter at several binding sites. Another crucial point to enhance the production of heterologous enzymes is the stability of recombinant mRNA and the prevention of protein degradation within the endoplasmic reticulum, especially for the bacteria originated enzymes.In this study, the CREI binding sites within the cbh1 promoter were replaced with the binding sites of transcription activator ACEII and the HAP2/3/5 complex to improve the promoter efficiency. To further improve heterologous expression efficiency of bacterial genes within T. reesei, a flexible polyglycine linker and a rigid α-helix linker were tested in the construction of fusion genes between cbh1 from T. reesei and e1, encoding an endoglucanase from Acidothermus cellulolyticus.ResultsThe modified promoter resulted in an increased expression level of the green fluorescent protein reporter by 5.5-fold in inducing culture medium and 7.4-fold in repressing culture medium. The fusion genes of cbh1 and e1 were successfully expressed in T. reesei under the control of promoter pcbh1m2. The higher enzyme activities and thermostability of the fusion protein with rigid linker indicated that the rigid linker might be more suitable for the heterologous expression system in T. reesei. Compared to the parent strain RC30-8, the FPase and CMCase activities of the secreted enzyme mixture from the corresponding transformant R1 with the rigid linker increased by 39% and 30% at 60°C, respectively, and the reduced sugar concentration in the hydrolysate of pretreated corn stover (PCS) was dramatically increased by 40% at 55°C and 169% at 60°C when its enzyme mixture was used in the hydrolysis.ConclusionsThis study shows that optimizations of the promoter and linker for hybrid genes can dramatically improve the efficiency of heterologous expression of cellulase genes in T. reesei.
| S-EPMC3342899 | biostudies-literature
Project description:Trichoderma reesei is an established protein production host with high natural capacity to secrete enzymes. The lack of efficient genome engineering approaches and absence of robust constitutive gene expression systems limits exploitation of this organism in some protein production applications. Here we report engineering of T. reesei for high-level production of highly enriched lipase B of Candida antarctica (calB) using glucose as a carbon source. Multiplexed CRISPR/Cas9 in combination with the use of our recently established synthetic expression system (SES) enabled accelerated construction of strains, which produced high amounts of highly pure calB. Using SES, calB production levels in cellulase-inducing medium were comparable to the levels obtained by using the commonly employed inducible cbh1 promoter, where a wide spectrum of native enzymes were co-produced. Due to highly constitutive expression provided by the SES, it was possible to carry out the production in cellulase-repressing glucose medium leading to around 4 grams per liter of fully functional calB and simultaneous elimination of unwanted background enzymes.
| S-EPMC6430808 | biostudies-literature
Project description:The cellulase and hemicellulase genes of the filamentous fungus Trichoderma reesei have been shown to be under carbon catabolite repression mediated by the regulatory gene cre1. In this study, strains were constructed in which the cre1 gene was either completely removed or replaced by a truncated mutant variant, cre1-1, found previously in the Rut-C30 mutant strain with enhanced enzyme production capability. The T. reesei transformants with either deletion or truncation of cre1 had clearly altered colony morphology compared with the parental strains, forming smaller colonies and fewer aerial hyphae and spores. Liquid cultures in a medium with glucose as a carbon source showed that the transformants were derepressed in cellulase and hemicellulase production. Interestingly, they also produced significantly elevated levels of these hydrolytic enzymes in fermentations carried out in a medium inducing the hydrolase genes. This suggests that cre1 acts as a modulator of cellulase and hemicellulase gene expression under both noninducing and inducing conditions. There was no phenotypic difference between the Deltacre1 and cre1-1 mutant strains in any of the experiments done, indicating that the cre1-1 gene is practically a null allele. The results of this work indicate that cre1 is a valid target gene in strain engineering for improved enzyme production in T. reesei.
| S-EPMC2708423 | biostudies-literature
Project description:Filamentous fungal strains of Trichoderma reesei have been widely used for cellulase production, and great effort has been devoted to enhancing their cellulase titers for the economic biorefinery of lignocellulosic biomass. In our previous studies, artificial zinc finger proteins (AZFPs) with the Gal4 effector domain were used to enhance cellulase biosynthesis in T. reesei, and it is of great interest to modify the AZFPs to further improve cellulase production. In this study, the endogenous activation domain from the transcription activator Xyr1 was used to replace the activation domain of Gal4 of the AZFP to explore impact on cellulase production. The cellulase producer T. reesei TU-6 was used as a host strain, and the engineered strains containing the Xyr1 and the Gal4 activation domains were named as T. reesei QS2 and T. reesei QS1, respectively. Compared to T. reesei QS1, activities of filter paper and endoglucanases in crude cellulase produced by T. reesei QS2 increased 24.6 and 50.4%, respectively. Real-time qPCR analysis also revealed significant up-regulation of major genes encoding cellulase in T. reesei QS2. Furthermore, the biomass hydrolytic performance of the cellulase was evaluated, and 83.8 and 97.9% more glucose was released during the hydrolysis of pretreated corn stover using crude enzyme produced by T. reesei QS2, when compared to the hydrolysis with cellulase produced by T. reesei QS1 and the parent strain T. reesei TU-6. As a result, we proved that the effector domain in the AZFPs can be optimized to construct more effective artificial transcription factors for engineering T. reesei to improve its cellulase production.
| S-EPMC7330100 | biostudies-literature
Project description:We overexpressed the err1 gene in the Trichoderma reesei wild-type and in the cellulase hyperproducing, carbon catabolite derepressed strain Rut-C30 in order to investigate the possibility of producing erythritol with T. reesei. Two different promoters were used for err1 overexpression in both strains, a constitutive (the native pyruvat kinase (pki) promoter) and an inducible one (the native ?-xylosidase (bxl1) promoter). The derived recombinant strains were precharacterized by analysis of err1 transcript formation on D-xylose and xylan. Based on this, one strain of each type was chosen for further investigation for erythritol production in shake flasks and in bioreactor experiments. For the latter, we used wheat straw pretreated by an alkaline organosolve process as lignocellulosic substrate. Shake flask experiments on D-xylose showed increased erythritol formation for both, the wild-type and the Rut-C30 overexpression strain compared to their respective parental strain. Bioreactor cultivations on wheat straw did not increase erythritol formation in the wild-type overexpression strain. However, err1 overexpression in Rut-C30 led to a clearly higher erythritol formation on wheat straw.
| S-EPMC4052684 | biostudies-literature
Project description:The filamentous fungus Trichoderma reesei is a widely used strain for cellulolytic enzyme production. A hypercellulolytic T. reesei variant SN1 was identified in this study and found to be different from the well-known cellulase producers QM9414 and RUT-C30. The cellulose-degrading enzymes of T. reesei SN1 show higher endoglucanase (EG) activity but lower ?-glucosidase (BGL) activity than those of the others. A uracil auxotroph strain, SP4, was constructed by pyr4 deletion in SN1 to improve transformation efficiency. The BGL1-encoding gene bgl1 under the control of a modified cbh1 promoter was overexpressed in SP4. A transformant, SPB2, with four additional copies of bgl1 exhibited a 17.1-fold increase in BGL activity and a 30.0% increase in filter paper activity. Saccharification of corncob residues with crude enzyme showed that the glucose yield of SPB2 is 65.0% higher than that of SP4. These results reveal the feasibility of strain improvement through the development of an efficient genetic transformation platform to construct a balanced cellulase system for biomass conversion.
| S-EPMC5002442 | biostudies-literature
Project description:Trichoderma reesei can produce up to 100 g/liter of extracellular proteins. The major and industrially relevant products are cellobiohydrolase I (CBHI) and the hemicellulase XYNI. The genes encoding both enzymes are transcriptionally activated by the regulatory protein Xyr1. The first 850 nucleotides of the cbh1 promoter contain 14 Xyr1-binding sites (XBS), and 8 XBS are present in the xyn1 promoter. Some of these XBS are arranged in tandem and others as inverted repeats. One such cis element, an inverted repeat, plays a crucial role in the inducibility of the xyn1 promoter. We investigated the impact of the properties of such cis elements by shuffling them by insertion, exchange, deletion, and rearrangement of cis elements in both the cbh1 and xyn1 promoter. A promoter-reporter assay using the Aspergillus nigergoxA gene allowed us to measure changes in the promoter strength and inducibility. Most strikingly, we found that an inverted repeat of XBS causes an important increase in cbh1 promoter strength and allows induction by xylan or wheat straw. Furthermore, evidence is provided that the distances of cis elements to the transcription start site have important influence on promoter activity. Our results suggest that the arrangement and distances of cis elements have large impacts on the strength of the cbh1 promoter, whereas the sheer number of XBS has only secondary importance. Ultimately, the biotechnologically important cbh1 promoter can be improved by cis element rearrangement.IMPORTANCE In the present study, we demonstrate that the arrangement of cis elements has a major impact on promoter strength and inducibility. We discovered an influence on promoter activity by the distances of cis elements to the transcription start site. Furthermore, we found that the configuration of cis elements has a greater effect on promoter strength than does the sheer number of transactivator binding sites present in the promoter. Altogether, the arrangement of cis elements is an important factor that should not be overlooked when enhancement of gene expression is desired.
| S-EPMC5734013 | biostudies-literature
Project description:BACKGROUND:The filamentous fungus Trichoderma reesei (teleomorph Hypocrea jecorina) is a widely used industrial host organism for protein production. In industrial cultivations, it can produce over 100 g/l of extracellular protein, mostly constituting of cellulases and hemicellulases. In order to improve protein production of T. reesei the transcriptional regulation of cellulases and secretory pathway factors have been extensively studied. However, the metabolism of T. reesei under protein production conditions has not received much attention. RESULTS:To understand the physiology and metabolism of T. reesei under protein production conditions we carried out a well-controlled bioreactor experiment with extensive analysis. We used minimal media to make the data amenable for modelling and three strain pairs to cover different protein production levels. With RNA-sequencing transcriptomics we detected the concentration of the carbon source as the most important determinant of the transcriptome. As the major transcriptional response concomitant to protein production we detected the induction of selected genes that were putatively regulated by xyr1 and were related to protein transport, amino acid metabolism and transcriptional regulation. We found novel metabolic responses such as production of glycerol and a cellotriose-like compound. We then used this cultivation data for flux balance analysis of T. reesei metabolism and demonstrate for the first time the use of genome wide stoichiometric metabolic modelling for T. reesei. We show that our model can predict protein production rate and provides novel insight into the metabolism of protein production. We also provide this unprecedented cultivation and transcriptomics data set for future modelling efforts. CONCLUSIONS:The use of stoichiometric modelling can open a novel path for the improvement of protein production in T. reesei. Based on this we propose sulphur assimilation as a major limiting factor of protein production. As an organism with exceptional protein production capabilities modelling of T. reesei can provide novel insight also to other less productive organisms.
| S-EPMC4924338 | biostudies-literature
Project description:BackgroundThe path for the development of hypersecreting strains of Trichoderma reesei capable of producing industrially relevant enzyme titers remains elusive despite over 70 years of research and industrial utilization. Herein, we describe the rational engineering of the publicly available T. reesei RUT-C30 strain and a customized process for cellulase production based on agroindustrial by-products.ResultsA CRISPR/Cas9 system was used to introduce six genetic modifications in RUT-C30. Implemented changes included the constitutive expression of a mutated allele of the cellulase master regulator XYR1, the expression of two heterologous enzymes, the β-glucosidase CEL3A from Talaromyces emersonii and the invertase SUC1 from Aspergillus niger, and the deletion of genes encoding the cellulase repressor ACE1 and the extracellular proteases SLP1 and PEP1. These alterations resulted in a remarkable increase of protein secretion rates by RUT-C30 and amended its well described β-glucosidase deficiency while enabling the utilization of sucrose and eliminating the requirement of inducing sugars for enzyme production. With a developed sugarcane molasses-based bioprocess, the engineered strain reached an extracellular protein titer of 80.6 g L-1 (0.24 g L-1 h-1), which is the highest experimentally supported titer so far reported for T. reesei. The produced enzyme cocktail displayed increased levels of cellulase and hemicellulase activities, with particularly large increments being observed for the specific activities of β-glucosidase (72-fold) and xylanase (42-fold). Notably, it also exhibited a saccharification efficiency similar to that of a commercially available cellulase preparation in the deconstruction of industrially pretreated sugarcane straw.ConclusionThis work demonstrates the rational steps for the development of a cellulase hyperproducing strain from a well-characterized genetic background available in the public domain, the RUT-C30, associated with an industrially relevant bioprocess, paving new perspectives for Trichoderma research on cellulase production.
| S-EPMC7243233 | biostudies-literature