Light-inducible genetic engineering and control of non-homologous end-joining in industrial eukaryotic microorganisms: LML 3.0 and OFN 1.0

Abstract

Filamentous fungi play important roles in the production of plant cell-wall degrading enzymes. In recent years, homologous recombinant technologies have contributed significantly to improved enzymes production and system design of genetically manipulated strains. When introducing multiple gene deletions, we need a robust and convenient way to control selectable marker genes, especially when only a limited number of markers are available in filamentous fungi. Integration after transformation is predominantly nonhomologous in most fungi other than yeast. Fungal strains deficient in the non-homologous end-joining (NHEJ) pathway have limitations associated with gene function analyses despite they are excellent recipient strains for gene targets. We describe strategies and methods to address these challenges above and leverage the power of resilient NHEJ deficiency strains. We have established a foolproof light-inducible platform for one-step unmarked genetic modification in industrial eukaryotic microorganisms designated as 'LML 3.0', and an on-off control protocol of NHEJ pathway called 'OFN 1.0', using a synthetic light-switchable transactivation to control Cre recombinase-based excision and inversion. The methods provide a one-step strategy to sequentially modify genes without introducing selectable markers and NHEJ-deficiency. The strategies can be used to manipulate many biological processes in a wide range of eukaryotic cells.

Figure 1. Construction of LML cassettes.

(A) LML 1.0 and 2.0 cassettes. (B) Multicolor labeling cassette LML 2.0s and fluorescence micrographs of marker self-excision course using fluorescence microscopy in H. jecorina transformed with LML 2.0s. Bars represent 20 μm.

Figure 2. Construction of LML 2.1, 2.11, 2.12 and 3.0 cassettes.

Figure 3. An on-off control protocol of nonhomologous end-joining (NHEJ) pathway using the Cre-lox system.

(A) Construction of OFN 1.0A–D cassettes. Genomic PCR of multiple strains using a given primer configuration (left bottom) shows inversion only after Cre induction. (B) Comparative transcript ratio analysis of tku70. Transcript ratios for ON and OFF state were calculated using ABI Stepone plus software. Values above 1 indicate higher transcription in the ON state strain compared to QM6a, and values below 1 indicate lower transcription. Error bars represent 95% confidence intervals. ***means not done.

Figure 4. Survival rate of of Qm6a, Qm6aΔtku70, Qm6a&OFN1.0D-ON and Qm6a&OFN1.0D-OFF strains following exposure to UV.

Spores were exposed to different doses of UV. Aliquots of the UV-irradiated spores were plated on potato dextrose agar plates containing the colony restrictor Triton X-100 and the surviving colonies were counted.

Figure 5

Relationship between LML 2.0/2.1/3.0 system and ZFNs (A), TALENs (B) or CRISPR (C) system.