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Modification of transcriptional factor ACE3 enhances protein production in Trichoderma reesei in the absence of cellulase gene inducer - PubMed

  • ️Wed Jan 01 2020

Modification of transcriptional factor ACE3 enhances protein production in Trichoderma reesei in the absence of cellulase gene inducer

Yun Luo et al. Biotechnol Biofuels. 2020.

Abstract

Background: Trichoderma reesei is one of the best-known cellulolytic organisms, producing large quantities of a complete set of extracellular cellulases and hemicellulases for the degradation of lignocellulosic substances. Hence, T. reesei is a biotechnically important host and it is used commercially in enzyme production, of both native and foreign origin. Many strategies for producing enzymes in T. reesei rely on the cbh1 and other cellulase gene promoters for high-level expression and these promoters require induction by sophorose, lactose or other inducers for high productivity during manufacturing.

Results: We described an approach for producing high levels of secreted proteins by overexpression of a transcription factor ACE3 in T. reesei. We refined the ace3 gene structure and identified specific ACE3 variants that enable production of secreted cellulases and hemicellulases on glucose as a sole carbon source (i.e., in the absence of an inducer). These specific ACE3 variants contain a full-length Zn2Cys6 binuclear cluster domain at the N-terminus and a defined length of truncations at the C-terminus. When expressed at a moderate level in the fungal cells, the ACE3 variants can induce high-level expression of cellulases and hemicellulases on glucose (i.e., in the absence of an inducer), and further improve expression on lactose or glucose/sophorose (i.e., in the presence of an inducer). Finally, we demonstrated that this method is applicable to industrial strains and fermentation conditions, improving protein production both in the absence and in the presence of an inducer.

Conclusions: This study demonstrates that overexpression of ACE3 variants enables a high level of protein production in the absence of an inducer, and boosts protein production in the presence of an inducer. It is an efficient approach to increase protein productivity and to reduce manufacturing costs.

Keywords: ACE3 transcription factor; Inducer-free; Trichoderma reesei.

© The Author(s) 2020.

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Conflict of interest statement

Competing interestsThe authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1

Gene structure and transcripts at the ace3 locus. a Schematic presentation of ace3 gene annotations at its native locus. Exons are shown as arrows and introns as single lines. In parenthesis are the origins of gene annotations. ▼, Transcriptional start site (TSS); dashed line, non-sense mutation; Cys: cysteine of the Zn2Cys6 binuclear domain. b Transcription levels of ace3 exons in strain QM9414 grown on different carbon sources for 24 h, as measured by RNA-seq. Normalized read counts are graphed using 100-bp bins per million mapped reads (BPM). c RT-qPCR analysis of expression levels of ace3 exons, Intron 2, and 5′UTR part of Intron 2. The 5′UTR is located within intron 2 and upstream of the ace3-SC ORF. The transcript expression levels are normalized to the house keeping gene act1, and are shown as relative ratios to the level of exon 1 at early growth phase. The mean ± standard deviation was calculated from three independent experiments

Fig. 2
Fig. 2

Overexpression of ace3 variants. a Schematic presentation of the different ace3 variants. Exons are shown as boxes and introns as single lines. The N-terminal and C-terminal amino acid residues are shown. C2 and C4 indicate the presence of 2 and 4 cysteines of the Zn2Cys6 binuclear domain within exons, respectively. b SDS-PAGE gel from culture supernatants. An equal volume of culture supernatant was loaded in each lane. c The total secreted protein concentrations as measured by Bradford assays. The mean ± standard deviation was calculated from three biological replicates. d MULac activities measured from the culture supernatants. The mean ± standard deviation was calculated from three biological replicates. Lac: lactose; Glu: glucose

Fig. 3
Fig. 3

ace3-LC variants with C-terminal truncations. a Total secreted protein concentrations as measured by Bradford assays. Lac: lactose; Glu: glucose. b Schematic representation of the ACE3-LC variant protein with a wild-type C-terminus. Exons are shown as boxes and introns as single lines. C2 and C4 indicate the presence of 2 and 4 cysteines of the Zn2Cys6 binuclear domain within exons, respectively. The first 3 amino acids at the N-terminus and the last 25 amino acids at the C-terminus are shown, and the amino acids at position -17 to -7 are underlined

Fig. 4
Fig. 4

ace3 deletion. a Schematic representation of the different ace3 variants. Exons are shown as boxes and introns as single lines. The N-terminal and C-terminal amino acid residues are shown. C2 and C4 indicate the presence of 2 and 4 cysteines of the Zn2Cys6 binuclear domain within exons, respectively. b SDS-PAGE gel from culture supernatants. An equal volume of culture supernatant was loaded in each lane. c Total secreted protein concentrations as measured by Bradford assays. The mean ± standard deviation was calculated from three biological replicates. d MULac activities measured from the culture supernatants. The mean ± standard deviation was calculated from three biological replicates. Lac: lactose; Glu: glucose

Fig. 5
Fig. 5

xyr1 deletion (a and b) and overexpression (c and d). a, c SDS-PAGE gel of culture supernatants. An equal volume of culture supernatant was loaded in each lane. b, d Total secreted protein concentrations as measured by Bradford assays. The mean ± standard deviation was calculated from three biological replicates. Lac: lactose; Glu: glucose

Fig. 6
Fig. 6

Overexpression of ace3-L improves protein production in an industrial strain. a SDS-PAGE of culture supernatants from T. reesei parental strain T4abc and its daughter strain LT83 (expressing ace3-L). An equal volume of culture supernatant was loaded in each lane. The total secreted protein concentration of each strain (as measured by Bradford assays) was shown as relative ratio to that of strain T4abc grown glucose/sophorose (set at 1). Glu/Sop, glucose/sophorose; Glu, glucose; M, protein molecular weight marker. b Protein production of T. reesei parental strain T4abc and daughter strain LT83 in 2-L fermenters. The total protein produced by the parental strain fed with glucose/sophorose at the end of fermentation was arbitrarily set at 1, and the relative amounts of protein produced by each strain at each time points were plotted

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