New tools for the genetic manipulation of filamentous fungi

Prepared by / Hussein Abu Ammar

Filamentous fungi in industry

The advent of DNA-mediated transformation procedures in the 1970s and 1980s led to alternative and more targeted strategies for the production of commercially valuable microbial strains, after that genetic engineering and genome wide sequencing are held .

Selecting a traditional filamentous fungi to improve a special genetically modified strains , mostly by random mutagenesis .

Filamentous fungi in industry

• Has important role as producers of primary and secondary metabolites with Biotechnical and pharmaceutical relevance ( cell factories for the production of chemicals, pharmaceuticals and enzymes).

Development mechanisms

1) Homologous DNA recombination .i. knock-in transformants.ii. knock-out transformants

1) RNA interference . I.knock-down transformants

1. Homologous DNA recombination

The homologous recombination is necessary in order to allow effective analysis of gene function. In principle, two types of transformants can be distinguished:

(1) knock-in transformants carry a foreign DNA sequence inserted in the target gene, resulting in truncated or aberrant transcripts. (2) in knock-out transformants, the target gene is substituted by a marker gene (e.g., resistance gene), and the target gene is therefore no longer expressed.

Homologous recombination in filamentous fungi usingstrains with a deficient non-homologous end

joining(NHEJ) One disadvantage still, the frequency of HR in

filamentous fungi is very low (usually between 0 and 30%).

One approach to overcome this limitation is using strains which are defective for the nonhomologous end joining pathway (NHEJ) .

No(NHEJ) will increase the frequency of homologous recombination .in other words, if the two breaks won’t be repaired the genetic material will be exchanged between two homologous chromosomes .

non-homologous end joining(NHEJ)

• NHEJ is mediated by a multi-protein complex containing the DNA-dependent protein kinase (DNA-PKcs), the DNA ligase IV-XRCC4 complex, the exonuclease Artemis, and the Ku70/Ku80 heterodimer that binds tightly to DNA ends and directs the DNA-PKcs, thereby allowing efficient activation.

2. RNA interference • RNA interference (RNAi) : RNA interference

(RNAi) is a biological process in which RNA molecules inhibit gene expression or translation, by neutralizing targeted mRNA molecules.

• “post-transcriptional gene silencing” -The silencing of gene expression by segments of DNA present in excess of the normal number is called cosuppression in plants and quelling in fungi.

RNA interference in eukaryotic

Meiotic silencing by unpaired DNA• Meiotic silencing by unpaired DNA (MSUD) :In Neurospora,

a gene not paired(Such unpairing events can be caused by gene deletions, duplications, and transpositions) with a homolog in prophase I of meiosis generates a signal that transiently silences all sequences homologous to it by a process called(MSUD).

• MSUD has so far only been described for filamentous fungi.• MSUD controls meiotic pairing and is important for the

correct gene expression during meiosis. Mutant strains from N. crassa carrying an ectopically integrated transgene whose expression occurs usually during meiosis will have aberrant meiosis.

MSUD• MSUD appears to involve the production of a single-stranded

aberrant RNA from an unpaired segment. This RNA is exported to the perinuclear region where it acts as a template for the SAD-1–mediated double-stranded (ds)RNA synthesis. DCL-1 then processes the dsRNA into small interfering (si)RNA, which guide SMS-2 to identify and slice complementary mRNA.

Vector systemsStem-loop expression :• Disadvantage : Time consuming to construct two opposite inversely

orientated target fragments to express double stranded RNA with a hairpin structure.

• Advantage : stable and high silencing frequencies.

Dual promotor :• single target fragment is transcribed from both sites by divergently

orientated fungal promoter sequences .• Advantage : vector easily be constructed.

Conclusions

1) Technical, pharmaceutical, or medical importance .

2) Novel application :Producers for new metabolites with an industrial

significance .Provide recombinant strains for industrial large

scale production processes.