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Induction of cellulase production in Trichoderma reesei by a glucose-sophorose mixture as an inducer prepared using stevioside.

ABSTRACT: Sophorose is currently the most effective inducer of cellulase production by Trichoderma reesei; however, the use of byproduct sophorose from the stevioside acid hydrolysis process has not been developed. In this study, stevioside was hydrolysed with different concentrations of HCl to obtain isosteviol and a mixture of glucose and sophorose (MGS). Isosteviol showed good inhibitory effects on the growth of Aspergillus niger, Saccharomyces cerevisiae and Escherichia coli after separation. At the same time, MGS, as a byproduct, was evaluated for cellulase production to determine the feasibility of this approach. MGS was compared with common soluble inducers, such as lactose, cellobiose, and a mixture of glucose and β-disaccharide (MGD), and induced higher cellulase production than the other inducers. The cellulase activity induced by MGS was 1.64- and 5.26-fold higher than that induced by lactose and cellobiose, respectively, and was not significantly different from that induced by MGD. The crude enzyme using MGS as an inducer with commercial β-glucosidase was further tested by hydrolyzing NaOH-pretreated corn stover with 5% solid loading, and 33.4 g L^-1 glucose was released with a glucose yield of 96.04%. The strategy developed in this work will be beneficial for reducing inducer production cost through a simple stevia glycoside hydrolysis reaction and will contribute to studies aimed at improving cellulase production using soluble inducers for easier operation in industrial-scale cellulase production.

SUBMITTER: Zhang P

PROVIDER: S-EPMC9190947 | biostudies-literature | 2022 Jun

REPOSITORIES: biostudies-literature

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Induction of cellulase production in Trichoderma reesei by a glucose-sophorose mixture as an inducer prepared using stevioside.

Zhang Peng P Li Qian Q Chen Yudian Y Peng Nian N Liu Wenshu W Wang Xuemei X Li Yonghao Y

RSC advances 20220613 27

Sophorose is currently the most effective inducer of cellulase production by Trichoderma reesei; however, the use of byproduct sophorose from the stevioside acid hydrolysis process has not been developed. In this study, stevioside was hydrolysed with different concentrations of HCl to obtain isosteviol and a mixture of glucose and sophorose (MGS). Isosteviol showed good inhibitory effects on the growth of Aspergillus niger, Saccharomyces cerevisiae and Escherichia coli</i ...[more]

PMID: 35765440

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Project description:BackgroundThe enzymes for efficient hydrolysis of lignocellulosic biomass are a major factor in the development of an economically feasible cellulose bioconversion process. Up to now, low hydrolysis efficiency and high production cost of cellulases remain the significant hurdles in this process. The aim of the present study was to develop a versatile cellulase system with the enhanced hydrolytic efficiency and the ability to synthesize powerful inducers by genetically engineering Trichoderma reesei.ResultsIn our study, we employed a systematic genetic strategy to construct the carbon catabolite-derepressed strain T. reesei SCB18 to produce the cellulase complex that exhibited a strong cellulolytic capacity for biomass saccharification and an extraordinary high β-glucosidase (BGL) activity for cellulase-inducing disaccharides synthesis. We first identified the hypercellulolytic and uracil auxotrophic strain T. reesei SP4 as carbon catabolite repressed, and then deleted the carbon catabolite repressor gene cre1 in the genome. We found that the deletion of cre1 with the selectable marker pyrG led to a 72.6% increase in total cellulase activity, but a slight reduction in saccharification efficiency. To facilitate the following genetic modification, the marker pyrG was successfully removed by homologous recombination based on resistance to 5-FOA. Furthermore, the Aspergillus niger BGLA-encoding gene bglA was overexpressed, and the generated strain T. reesei SCB18 exhibited a 29.8% increase in total cellulase activity and a 51.3-fold enhancement in BGL activity (up to 103.9 IU/mL). We observed that the cellulase system of SCB18 showed significantly higher saccharification efficiency toward differently pretreated corncob residues than the control strains SDC11 and SP4. Moreover, the crude enzyme preparation from SCB18 with high BGL activity possessed strong transglycosylation ability to synthesize β-disaccharides from glucose. The transglycosylation product was finally utilized as the inducer for cellulase production, which provided a 63.0% increase in total cellulase activity compared to the frequently used soluble inducer, lactose.ConclusionsIn summary, we constructed a versatile cellulase system in T. reesei for efficient biomass saccharification and powerful cellulase inducer synthesis by combinational genetic manipulation of three distinct types of genes to achieve the customized cellulase production, thus providing a viable strategy for further strain improvement to reduce the cost of biomass-based biofuel production.

Project description:BackgroundThe filamentous fungus Trichoderma reesei is the main industrial cellulolytic enzyme producer. Several strains have been developed in the past using random mutagenesis, and despite impressive performance enhancements, the pressure for low-cost cellulases has stimulated continuous research in the field. In this context, comparative study of the lower and higher producer strains obtained through random mutagenesis using systems biology tools (genome and transcriptome sequencing) can shed light on the mechanisms of cellulase production and help identify genes linked to performance. Previously, our group published comparative genome sequencing of the lower and higher producer strains NG 14 and RUT C30. In this follow-up work, we examine how these mutations affect phenotype as regards the transcriptome and cultivation behaviour.ResultsWe performed kinetic transcriptome analysis of the NG 14 and RUT C30 strains of early enzyme production induced by lactose using bioreactor cultivations close to an industrial cultivation regime. RUT C30 exhibited both earlier onset of protein production (3 h) and higher steady-state productivity. A rather small number of genes compared to previous studies were regulated (568), most of them being specific to the NG 14 strain (319). Clustering analysis highlighted similar behaviour for some functional categories and allowed us to distinguish between induction-related genes and productivity-related genes. Cross-comparison of our transcriptome data with previously identified mutations revealed that most genes from our dataset have not been mutated. Interestingly, the few mutated genes belong to the same clusters, suggesting that these clusters contain genes playing a role in strain performance.ConclusionsThis is the first kinetic analysis of a transcriptomic study carried out under conditions approaching industrial ones with two related strains of T. reesei showing distinctive cultivation behaviour. Our study sheds some light on some of the events occurring in these strains following induction by lactose. The fact that few regulated genes have been affected by mutagenesis suggests that the induction mechanism is essentially intact compared to that for the wild-type isolate QM6a and might be engineered for further improvement of T. reesei. Genes from two specific clusters might be potential targets for such genetic engineering.

Project description:BackgroundThe filamentous fungus Trichoderma reesei (anamorph of Hypocrea jecorina) produces increased cellulase expression when grown on cellulose or its derivatives as a sole carbon source. It has been believed that β-glucosidases of T. reesei not only metabolize cellobiose but also contribute in the production of inducers of cellulase gene expression by their transglycosylation activity. The cellulase hyper-producing mutant PC-3-7 developed in Japan has enhanced cellulase production ability when cellobiose is used as the inducer. The comparative genomics analysis of PC-3-7 and its parent revealed a single-nucleotide mutation within the bgl2 gene encoding intracellular β-glucosidase II (BGLII/Cel1a), giving rise to an amino acid substitution in PC-3-7, which could potentially account for the enhanced cellulase expression when these strains are cultivated on cellulose and cellobiose.ResultsTo analyze the effects of the BGLII mutation in cellulase induction, we constructed both a bgl2 revertant and a disruptant. Enzymatic analysis of the transformant lysates showed that the strain expressing mutant BGLII exhibited weakened cellobiose hydrolytic activity, but produced some transglycosylation products, suggesting that the SNP in bgl2 strongly diminished cellobiase activity, but did not result in complete loss of function of BGLII. The analysis of the recombinant BGLII revealed that transglycosylation products might be oligosaccharides, composed probably of glucose linked β-1,4, β-1,3, or a mixture of both. PC-3-7 revertants of bgl2 exhibited reduced expression and inducibility of cellulase during growth on cellulose and cellobiose substrates. Furthermore, the effect of this bgl2 mutation was reproduced in the common strain QM9414 in which the transformants showed cellulase production comparable to that of PC-3-7.ConclusionWe conclude that BGLII plays an important role in cellulase induction in T. reesei and that the bgl2 mutation in PC-3-7 brought about enhanced cellulase expression on cellobiose. The results of the investigation using PC-3-7 suggested that other mutation(s) in PC-3-7 could also contribute to cellulase induction. Further investigation is essential to unravel the mechanism responsible for cellulase induction in T. reesei.

Dataset Information

Induction of cellulase production in Trichoderma reesei by a glucose-sophorose mixture as an inducer prepared using stevioside.

Publications

Induction of cellulase production in <i>Trichoderma reesei</i> by a glucose-sophorose mixture as an inducer prepared using stevioside.

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