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

Lei Zhang,Xihua Zhao,Dongzhi Wei,Suping Zhang,Guoxiu Zhang,Xuedong Wang,Jiajia Zhang,Wei Wang

doi:10.1038/srep20761

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.

Highlights

Filamentous fungi play important roles in the production of plant cell-wall degrading enzymes
Several methods have been reported for unmarked genetic modification of eukaryotic microorganisms
To prevent the bacterial expression of cre driven by fungal promoters, we initially constructed three chimeric cre genes (z1cre, z2cre and z3cre)

Summary

Introduction

Filamentous fungi play important roles in the production of plant cell-wall degrading enzymes. Homologous recombinant technologies have contributed significantly to improved enzymes production and system design of genetically manipulated strains. Recombinant technologies have contributed significantly to improved enzymes production and system design of genetically manipulated strains. Retransformation with a cre gene that remained in the fungal genome[3,4] is associated with the risk of chromosomal rearrangement and Cre toxicity[5] Another method is transient expression of Cre recombinase by Florea et al.[6]. A fourth option is PCR-based fusion of self-excision cassette, which includes a series of line DNA fragments fused by PCR, and PCR-generated errors might be introduced into the genome[8] Another potential disadvantage of this method is that the gene disruption cassettes they generated are linear DNA fragments by PCR. Light is an ideal inducer of gene expression because it is highly tunable and has high spatiotemporal resolution[11,12,13]

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