Fungal Transformation - dna transformation in fungi

Fungal Transformation

Fungal Transformation

Transformation in Fungi Yeast & Filamentous fungi

Presented by Abhishek GiriM.Sc. (Part II), SEM IV, P - VIR.K.Talreja College UNR-3

Topics to be Covered

Introduction to Fungi

Background of fungal transformation

Transformation protocol

Transformation vectors

Integration into chromosomes

Biological applications of fungi

Conclusion

References

Introduction

Fungi Eukaryotes, classified as yeasts or Filamentous fungi based on the form of growth in culture. Some are Dimorphic.

Fungi range in size from microscopic to macroscopic (e.g. mushroom) forms. Microscopic fungi include yeasts (usually unicellular) and filamentous fungi (e.g. molds).

Fungi have defined membrane-bound nuclei containing several chromosomes & ER.

Cell walls composed of various polysaccharides & glycoproteins depending upon the species.

Complex life cycle, vegetative growth as mycelium followed by morphogenesis with either sexual or asexual spores.

Fungi contain a larger genome (>10Mb compared to 4.7 Mb for E.coli) because fungi have more genes, & more non-coding DNA.

Yeast

Single cells of typically 5-10 m either spherical, cylindrical or oval.

Can grow well on a minimal medium containing D-glucose (also referred to as dextrose in food industry) as a C source and salts that supply N, P and trace metals. Under optimal growth conditions, doubling time = 90 min.

Can reproduce by asexual or sexual means.

Asexual means by Budding, fission.

Sexual mean involves formation of Zygote.

Yeast DNA is located within the mucleus & the modification of mRNA is similar to that of higher eukaryotes.

Filamentous fungi

Have a mycelial structure, a highly branched system of tubes, that contains mobile cytoplasm with many nuclei. A single long thin filament on the mycelium is called a hypha (plural: hyphae).

When grown in submerged culture, molds often form cell aggregates and pellets.

Molds are used for the production of citric acid (e.g. Aspergillus niger) and many antibiotics (e.g. Penicillium chrysogenum).

2when a conidia spore lands on a suitable substrate, it germinates and develops into hyphaeThe yeast Saccharomyces cerevisiae.

The yeast & hyphal forms of Yarrowia lipolytica.

Filamentous fungi Aspergillus niger grown on a cellophane sheet

Protoplast formation from Aspergillus nidulans.

First fungal transformation experiment was performed in 1970s.

Successful transformation relied on converting the fungal cells into protoplast by digesting the cell wall with carbohydrase enzyme before introducing the transforming DNA.

Transformants are then selected by their ability to grow without supplementation , a process known as genetic complementation.

Fate of transforming DNA it integrates with fungal chromosome.

Subsequent, transformation methods were developed for the yeast Saccharomyces cerevisiae using shuttle vectors as intact plasmids, without chromosomal integration.

Similar vectors have been designed for Filamentous fungi but in most cases the DNA integrates into the chromosome rather than replicating as a plasmid.

Transformation was first achieved in S. cerevisiae, Neurospora crassa, Aspergillus nidulans.

Background

Transformation Protocol

For transformation of many Filamentous fungi, the fungal mycelium is converted into protoplasts, prepared in a buffer containing a osmotic stabiliser (NaCl, MgS, mannitol, sorbitol) to prevent bursting & can be frozen at -70C for later use.

Vector DNA is then added to the protoplast suspension in the presence of Ca2+ ions & DNA uptake is induced by adding PEG.

The enzyme mixture marketed as Novozyme 234 is used.

The LiAc procedure, avoids protoplast formation used commonly for S. cerevisiae has been applied to N. crassa but has not found widespread use.

Transformation Protocol
(filamentous fungi)

Prepare the recombinant DNA.

Grow the cells, remove the cell walls by carbohydrase enzyme.

Wash protoplast with buffer containing the osmotic stabilizer.

Add plasmid DNA, CaCl2 & PEG to the cells.

Select the colonies that contain the foreign genes.

This protocol also applies to some yeasts such as S. cerevisiae.

However, yeast can be commonly transformed with Lithium acetate, provides a high transformation efficiency of 105 to 106 transformants per g DNA.

Fungal transformation methods

Transformation vectors

Can be designed to introduce DNA which either integrates into the genomic DNA (for most filamentous fungi) or can be maintained as a plasmid (for some yeasts).

Yeats shuttle vectors contain genes which allow for their selection in both bacterial & yeast cells, also contain ori, sequence.

To find the cells which are transformed. The vector is designed to contain a selection marker.

These markers fall into 3 main groups

Three groups of selectable markers:

Genes with antibiotics resistance, e.g. hygromycin, kanamycin, phleomycin etc. the drawback is that the fungus must be sensitive to the antibiotic.

Genes that can complement auxotrophic growth requirements. Many of the yeast markers encode functions that are involved in biosynthesis pathways of yeast, e.g. URA3 gene essential for uracil synthesis can complement ura3- mutants so these vectors must be transformed into the auxotrophic mutants.

Genes that confer the ability to grow on C or N sources which the host strain would not normally be able to use. e.g. amdS of A. nidulans which allows growth on acetamide or acrylamide as sole N source

A stylised yeast - E. coli shuttle vector

Shows ori, selection markers & cloning site A for inserting the gene to be expressed.

Site B is a restriction site required to convert the vector from a circular molecule to a linear one which increases the efficiency of DNA integration by homologous recombination into the rDNA region of the region.

Plasmid vectors are maintained provided the transformants are grown under selective pressure. Once the selective pressure is removed, the plasmids could be lost during the cell division.

Plasmid vectors can replicate with ori, an ori from one yeast strain can normally function in different yeast hosts, albeit not always with the same degree of efficiency. Up to 200 copies can be present in a single cell via additional selection.

Disadvantage maintaining the desired characteristic particularly in S. cerevisiae

However in other yeast such as Kluyveromyces lactis, some extra chromosomal vectors are comparatively stable.

Integration into chromosomes

Leads to enhanced stability, but lower numbers of introduced gene.

One example to enhance the number of genes in S. cerevisiae is to integrate into ribosomal DNA sequences which can be present at about 150 tandem repeats per genome.

The number of gene copies can be increased by placing the gene under the transcriptional control of weak, or deliberately weakened, promoters.

Integration can also be used to disrupt or replace a desired gene, which can be exploited to test the function of each gene in the cell.

Plasmid can survive in the yeast but typically foreign genes must be integrated into the filamentous fungi.

One approach to increase transformation is the use of REMI.

Restriction enzyme cut once in the vector . under these conditions, the DNA is targeted to the corresponding restriction sites in the genome.

with REMI the proportion of single copies of vector at several different sites is increased.

The extent of protein production is affected by the site of integration in the hosts genome.

Therefore, methods have been developed to target genes to specific locations that ensure good expression.

Biological Application of fungi

conclusion

The development of methods for transforming filamentous fungi have revolutionized all aspects of fungal research.

It has opened up molecular-genetic studies in previously difficult species.

Applications to fungal biotechnology are widespread.

Transformation is now being used to attack diverse problems in fungal biology.

Many other problems in fungal development can now be approached using such methods.

Increasingly sophisticated techniques for inactivating genes, targeting in vitro generated mutations to specific loci, & altering gene expression & its regulation are being developed to investigate the wealth of basic & applied biological problems available in filamentous fungi.

References- Books

Colin Rateledge and Bjorn Kristiansen, Basic Biotechnology, 2nd Edition, Cambridge Univ. Press.

Bernard R. Glick and Jack J. Pasternak, Molecular Biotechnology Principles and applications of recombinant DNA, ASM Press, Washington DC.

S. S. Purohit, Biotechnology Fundamentals and applications, 3rd Edition, Agro bios, India

References-Web links

www.eolss.net/sample-chapters/c17/e6-58-03-02.pdf

ebooks.cambridge.org/chapter.jsf?bid=CBO9780511802409&cid

www.nature.com/protocolexchange/protocols/427

2013.igem.org/Team:Cornell/project/.../fungal.../fungal_transformation

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Fungal Transformation - dna transformation in fungi

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