Transformation system for Hypocrea jecorina (Trichoderma reesei) that favors homologous integration and employs reusable bidirectionally selectable markers

Abstract

Hypocrea jecorina is an industrially important filamentous fungus due to its effective production of hydrolytic enzymes. It has received increasing interest because of its ability to convert lignocellulosic biomass to monomeric sugars, which can be converted into biofuels or platform chemicals. Genetic engineering of strains is a highly important means of meeting the requirements of tailor-made applications. Therefore, we report the development of a transformation system that allows highly efficient gene targeting by using a tmus53 (human LIG4 homolog) deletion strain. Moreover, it permits the unlimited reuse of the same marker by employing a Cre/loxP-based excision system. Both marker insertion and marker excision can be positively selected for by combining resistance to hygromycin B and loss of sensitivity to fluoroacetamide. Finally, the marker pyr4, also positively selectable for insertion and loss, can be used to remove the cre gene.

FIG. 1.

Construction of the vector bearing the loxP sites and the bidirectional marker system. (A) Amplification of the tmus53 5′-flanking region (light gray) and 3′-flanking region (dark gray) introducing a loxP site (black triangle) between the two regions. The first PCR (PCR1) yielded 1.4- and 1.3-kb products of the 5′- and 3′-flanking regions, including a loxP site on their 3′ end or 5′ end, respectively. A second PCR (PCR2) using the outer primers yielded an amplicon consisting of the 5′-flanking region of tmus53, a loxP site, and the 3′-flanking region of tmus53. Primer sequences are given in Table S1 in the supplemental material. Thin black arrows (⇀) indicate primers; thin black arrows with a black box indicate primers introducing a loxP site. sc5 means scaffold 5, and the genomic position is indicated. (B) Subcloning and Cre recombinase reaction. The obtained PCR product was subcloned into pGEM-T to obtain pMS-5loxP3-tmus, which was used as the acceptor vector in the subsequent Cre recombinase reaction. pMS-HALS, bearing two loxP sites flanking the amdS and hph genes as markers for fungal transformation and sacB as a killer gene (black segment), was used as the donor vector. The Cre recombinase reaction yielded pMS-loxPΔtmus53, bearing the tmus53 5′-flanking region, a loxP site, amdS, hph, a loxP site, and the tmus53 3′-flanking region. Short black lines indicate NotI sites; white arrows indicate enzymatic reactions.

FIG. 2.

Schematic drawing of strain construction and marker reuse. (A) Construction of the H. jecorina tmus53 deletion strain. The tmus53 gene was replaced by the bar gene conferring phosphinothricin resistance (P⁺) to yield strain QM6aΔtmus53. The boxes containing a checkerboard pattern or crosses indicate 5′-flanking region or 3′-flanking region of the tmus53 gene, respectively. sc5 means scaffold 5, and the genomic position is indicated. White arrows indicate a fungal transformation step. (B) Deletion of the pyr4 gene and replacement of phosphinothricin resistance by a bidirectional marker system. In a cotransformation, the bar gene was replaced by the loxP-amdS-hph cassette conferring hygromycin B resistance (H⁺) and the ability to use acetamide as nitrogen source (A⁺), and the pyr4 gene was replaced by the cre gene encoding Cre recombinase to yield strain QM6aΔtmus53Δpyr4. The boxes containing a grid pattern or white circles linked together on a black background indicate the 5′-flanking region or the 3′-flanking region of the pyr4 gene, respectively. (C) Marker excision. Growing the QM6aΔtmus53Δpyr4 strains on xylan (1% [wt/vol]) for 3 days promoted excision of the marker genes (amdS and hph) via the flanking loxP sites. (D) Two options for further manipulations. The first option is back transformation of the pyr4 gene replacing the cre gene to obtain a marker-free QM6aΔtmus53 strain. The second option is another round of deletion of any gene (xxx) (in the present study the PAS domain of env1) to obtain QM6aΔtmus53Δpyr4Δxxx, which is hygromycin B resistant (H⁺) and able to use acetamide as a nitrogen source (A⁺). The boxes containing a brick pattern or white circles linked on a black background indicate 5′-flanking region or 3′-flanking region of the env1 PAS domain, respectively. The two black arrows to the left indicate the two options.

FIG. 3.

Schematic drawing of the primer positions for analytic PCR of the loci. Small black arrows (⇀) indicate the locations of primers that were used for analyses of transformants and parental strains. The size of the expected amplicon is given between the broken lines in kilobases. The boxes containing a grid pattern or white linked circles on a black background indicate the 5′-flanking region or the 3′-flanking region of the pyr4 gene, respectively, in panels A and B. The boxes containing squares or crosses indicate the 5′-flanking region or the 3′-flanking region of the tmus53 gene, respectively, in panels C to F. The boxes containing a brick pattern or white circles indicate the 5′-flanking region or the 3′-flanking region of the env1 PAS domain, respectively, in panel G. The corresponding sequences are given in the supplemental material (see sections A to G of Supplemental Data File S2).

FIG. 4.

Flow chart of fungal transformation steps and the corresponding strains obtained from H. jecorina QM6a. The strain genotypes are given in the strain designations in the boxes, and their phenotypes are given in parentheses. H⁺, hygromycin B resistance; A⁺, ability to use acetamide as a nitrogen source; loxP, loxP site(s); xxx, any gene to be deleted from the genome.