Development of genetic tools for Myceliophthora thermophila

Abstract

Background: The thermophilic filamentous fungus Myceliophthora thermophila has many suitable characteristics for industrial biotechnology and could be a promising new chassis system for synthetic biology, particularly the ATCC 42464 strain, whose genome was sequenced in 2011. However, metabolic engineering of this strain using genetic approaches has not been reported owing to a lack of genetic tools for this organism.

Results: In the present study, we developed a high efficiency Agrobacterium tumefaciens mediated transformation system for M. thermophila, including an approach for targeted gene deletion using green fluorescence protein (GFP) as a marker for selection. Up to 145 transformants per 10(5) conidia were obtained in one transformation plate. Moreover, a ku70 deletion mutant was constructed in the ATCC 42464 background using the tools developed in present study and subsequently characterized. The ku70 deletion construct was designed using resistance to phosphinothricin as the selection marker. Additionally, a GFP-encoding cassette was incorporated that allowed for the selection of site-specific (no fluorescence) or ectopic (fluorescence) integration of the ku70 construct. Transformants with ectopically integrated ku70 deletion constructs were therefore identified using the fluorescent signal of GFP. PCR and Southern blotting analyses of non-fluorescent putative ku70 deletion transformants revealed all 11 tested transformants to be correct deletions. The deletion frequency in a pool of 116 transformants analyzed was 58 %. Moreover, the homologous rate improved about 3 folds under ku70 mutant using the pyrG as a test gene to disrupt in M. thermophila.

Conclusions: We successfully developed an efficient transformation and target gene disruption approach for M. thermophila ATCC 42464 mediated by A. tumefaciens. The tools and the ku70 deletion strain developed here should advance the development of M. thermophila as an industrial host through metabolic engineering and accelerate the elucidation of the mechanism of rapid cellulose degradation in this thermophilic fungus.

Fig. 1

Confocal fluorescence imaging of mycelia (Top) and conidia (Bottom) of M. thermophila pPK2BarGFPD transformants

Fig. 2

Optimization of A. tumefaciens-mediated M. thermophila transformation. a The impact of acetosyringone (AS) on A. tumefaciens-mediated transformation efficiency. Co-cultivation was conducted in induction medium containing AS on indicated concentrations for 2 days. b The effect of co-culture time on transformation efficiency. Co-cultivation was conducted in induction medium containing 200 μM AS for various times indicated. Error bars indicate the standard deviation of three independent experiments

Fig. 3

Vector map of the pPK2-ku70 vector constructed based on binary vector pPK2BarGFPD. Kan, kanamycin resistance gene; Ptef, promoter of translation elongation factor gene from Aureobasidium pullulans; egfp, enhanced green fluorescence protein; PtrpC, promoter of tryptophan synthetase gene from Aspergillus nidulans; bar, phosphinothricin resistance gene; 3′-flank and 5′-flank, 3′ and 5′ flanking fragments of ku70, respectively; RB and LB, right and left border of T-DNA, respectively

Fig. 4

ku70 disruption of wild-type M. thermophila ATCC 42464. a Pattern of the ku70 knockout cassette integrating into the chromosomes of M. thermophila ATCC 42464 via ectopic insertion or homologous recombination. b PCR analysis of ku70 deletion transformants with one primer (ku70KO-F) located in the bar gene cassette and the other (ku70KO-R) located in the downstream of 3′ flank in the genomic DNA (expected product of 1872 bp) and c Southern blotting analysis of ku70 mutants with XhoI digested genomic DNA and the probe amplified from the 3′ flank. Lines 1–11 in (b) and (c), genomic DNA from ku70 mutants (expected product of 5056 bp). WT, genomic DNA from wild-type as negative control (expected product of 1703 bp). Primers are represented by solid black arrows. Abbreviations: gDNA, genomic DNA; RB and LB, right and left border of T-DNA, respectively

Fig. 5

Phenotypic characterization of ku70 mutant. a Protein concentration in supernatants from wild-type (WT) and ku70 mutant cultures grown on 1 % Avicel for 3 d at 45 °C. b SDS-PAGE gel of wild-type and Δku70 strains grown at 45 °C for 3 d on 1 % Avicel. c Conidiation of wild-type and Δku70 strains growth at 45 °C for 9 d on agar plate without selective agent. d The hyphae of wild-type and Δku70 strains growth at 45 °C for 24 h on liquid MM without selective agent

Fig. 6

pyrG gene deletion in wild type and ∆ku70 mutant of M. thermophila ATCC 42464. The experiment design (a), PCR analysis of transformants under wild-type background (b) and ∆ku70 mutant (c) with one primer (pyrGout-F) located in the neo gene cassette and the other (pyrGout-R) located in the downstream of 3′ flank in the genomic DNA. The genomic DNA from the wild-type (WT) and ∆ku70 strains was used as the negative control