Fungal Transformation in yeast and filamentous fungi Introduction to Fungi Background of fungal transformation Transformation protocol Transformation vectors Integration into chromosomes Biological applications of fungi Conclusion References

1. Fungal Transformation
Transformation in Fungi – Yeast & Filamentous fungi
Presented by – Abhishek Giri
M.Sc. (Part – II), SEM – IV, P - VI
R.K.Talreja College UNR-3

2. Topics to be Covered
O Introduction to Fungi
O Background of fungal transformation
O Transformation protocol
O Transformation vectors
O Integration into chromosomes
O Biological applications of fungi
O Conclusion
O References

3. 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.
•
•

4. 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.

5. 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).
when a conidia spore
lands on a suitable
substrate, it germinates
and develops into
hyphae

6. a) The yeast Saccharomyces
cerevisiae.
b) The yeast & hyphal forms of
Yarrowia lipolytica.
c) Filamentous fungi
Aspergillus niger grown
on a cellophane sheet
d) Protoplast formation from
Aspergillus nidulans.

7. • 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.
•
BackgroundBackground

8. 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 -70°C 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.
•

9. 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

10. Fungal transformation methods

11. 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
•

12. v 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
•

13. 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.

14. • 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.

15. 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.

16. • 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 host’s genome.
• Therefore, methods have been developed to target genes to
specific locations that ensure good expression.
•
•

17. Biological Application of fungi

18. 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.

19. References-Books
O Colin Rateledge and Bjorn Kristiansen, Basic
Biotechnology, 2nd Edition, Cambridge Univ.
Press.
O
O Bernard R. Glick and Jack J. Pasternak,
Molecular Biotechnology – Principles and
applications of recombinant DNA, ASM Press,
Washington DC.
O
O S. S. Purohit, Biotechnology – Fundamentals
and applications, 3rd Edition, Agro bios, India

20. References-Weblinks
O www.eolss.net/sample-chapters/c17/e6-58-03-02.pdf
O
O ebooks.cambridge.org/chapter.jsf?
bid=CBO9780511802409&cid
O
O www.nature.com/protocolexchange/protocols/427
O
O 2013.igem.org/Team:Cornell/project/.../fu
ngal.../fungal_transformation
O