Engineering of Trichoderma reesei for enhanced degradation of lignocellulosic biomass by truncation of the cellulase activator ACE3

Abstract

Background: The filamentous fungus Trichoderma reesei is a major workhorse employed to produce cellulase, which hydrolyzes lignocellulosic biomass for the production of cellulosic ethanol and bio-based products. However, the economic efficiency of biorefineries is still low.

Results: In this study, the truncation of cellulase activator ACE3 was identified and characterized in T. reesei classical mutant NG14 and its direct descendants for the first time. We demonstrated that the truncated ACE3 is the crucial cause of cellulase hyper-production in T. reesei NG14 branch. Replacing the native ACE3 with truncated ACE3 in other T. reesei strains remarkably improves cellulase production. By truncating ACE3, we engineered a T. reesei mutant, PC-3-7-A723, capable of producing more cellulase than other strains. In a 30-L fermenter, fed-batch fermentation with PC-3-7-A723 drastically increased the maximum cellulase titer (FPase) to 102.63 IU/mL at 240 h, which constitutes a 20-30% improvement to that of the parental strain PC-3-7.

Conclusions: This work characterized the function of truncated ACE3 and demonstrated that analysis of classical mutants allows rational engineering of mutant strains with improved cellulase production necessary to process lignocellulosic biomass. Our rational engineering strategy might be useful for enhancing the production of other bio-based products.

Keywords: Cellulase production; Genetic engineering; Lignocellulosic biomass; Trichoderma reesei; Truncated ACE3.

Fig. 1

Native and truncated ACE3 sequences and genealogy of strains used in this study. a Native and truncated ace3 DNA sequences. Red color represents the missense mutation loci in the sequence of ace3. Blue color represents the DNA sequence of the 11 truncated amino acids. The underlines indicate the stop codons. b Native type ACE3-734 and truncated type ACE3-723. Native ACE3 is composed of 734 amino acids, and truncated ACE3-723 is composed of 723 amino acids. c Genealogy of strains used in this study. Mutagens used appear next to strain names. The gray color used for the M7 strain indicates that the strain is no longer available and was not included in this study. The number 723 denoted in red represents the strain containing truncated ACE3-723. LA Linear accelerator; UV ultraviolet light; NTG N-nitroguanidine

Fig. 2

Construction of transformants and effects of the truncated type ACE3-723 versus the native type ACE3-734 on cellulase production of T. reesei Rut-C30. a Schematic representation of A723 and A734 transformants. LML 2.1 is the erasable hygromycin selection marker in T. reesei. A734 transformants carry the native ACE3-734 as the test strains. A723 transformants bear ACE3-723 as controls. The black square denotes the loxP site left at the C-terminus of ACE3 after the marker was excised. The primers ace3-CF and D70-4 and HG3.6 and ace3-CR were used to verify the genotype of ACE3. b, c Effects of the truncated type ACE3-723 versus the native type ACE3-734 on cellulase production of T. reesei. The pNPCase activity of T. reesei Rut-C30 and transformants was examined after culture in liquid 2× Mandels’ medium with 2% lactose (b) or 1% Avicel (c). d–f Transcription of genes encoding the major cellulase (cbh1) and essential transcription factors for cellulase (ace3 and xyr1) was evaluated. Three independent experiments with three biological replicates each were performed. The sar1 gene was used as the internal control for normalization. Values are the mean ± SD of the results from three independent experiments. Asterisks indicate a significant difference (*p < 0.05, Student’s t test)

Fig. 3

Effects of the truncated type ACE3-723 versus the native type ACE3-734 on cellulase production of T. reesei NG14 and RL-P37. The pNPCase activity of T. reesei NG14 (a, b) and RL-P37 (c, d) was examined after culture in liquid 2× Mandels’ medium with 2% lactose (a, c) or 1% Avicel (b, d). Values are the mean ± SD of results from three independent experiments. Asterisks indicate a significant difference (*p < 0.05, Student’s t test)

Fig. 4

Effects of the truncated type ACE3-723 versus the native type ACE3-734 on cellulase production in T. reesei QM6a, QM9414, and PC-3-7. The pNPCase activity of T. reesei QM6a (a, b), QM9414 (c, d), and PC-3-7 (e) was examined after culture in liquid 2× Mandels’ medium with 2% lactose (a, c) or 1% Avicel (b, d, e). Values are the mean ± SD of the results from three independent experiments. Asterisks indicate a significant difference (*p < 0.05, Student’s t test)

Fig. 5

Time-course of the fed-batch culture of T. reesei PC-3-7-A723 for cellulase production in a 30-L fermenter. Fermentation was started with MGDS feeding. The samples were taken at regular intervals, and the supernatant was analyzed for the FPase, pNPCase, pNPGase, and CMCase activities. Mycelia were collected for biomass measurement. Values are the mean ± SD of results from three triplicate measurements

Fig. 6

Schematic diagrams for producing cellulosic ethanol by genetic engineering of filamentous fungi T. reesei PC-3-7-A723. The truncated type ACE3-723 is transformed into T. reesei PC-3-7 to generate the engineered cellulase hyper-producer PC-3-7-A723. The truncated ACE3-723 induces cellulase expression by interacting with XYR1. PC-3-7-A723 inherits the classical mutation sites in CRE1 (releasing CCR to enhance cellulase gene expression) [22], BGLR (decreasing β-glucosidase gene expression and increasing cellulase gene expression) [24], and BGL2 (promoting the conversion of cellobiose to sophorose to induce cellulase gene expression) [13] from PC–3–7, which contribute to the improvements of cellulase. We integrated these crucial mutations in PC-3-7-A723 by rational engineering to promote the cellulase production for cellulosic ethanol