Chap 5 Genetic Engineering: yeast and filamentous fungi
I. Introduction

Fungi range in size from microscopic to macroscopic (e.g. mushroom) forms.
Microscopic fungi include yeasts (usually unicellular) and filamentous (細絲狀) fungi
(e.g. molds 黴菌).
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.
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Fungi contain a larger genome (>10 Mb compared to 4.7 Mb for E. coli) because fungi
have more genes, and more non-coding DNA.
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Fungal cell walls do not contain peptidoglycan which is found only in bacteria. Rather,
their walls are composed primarily of
Yeasts: (See Shuler Chap. 2 and Ausubel Chap 13)
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Single cells of typically 5-10 m (but can vary from 2-3 m to 20-50 m). Either
spherical, cylindrical or oval.
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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.
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Can reproduce by asexual or sexual means.
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Asexual reproduction:
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budding: a small bud1 forms on the cell, which gradually enlarges and separates
from the mother cell.
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fission: similar to that of bacteria. In fission, cells grow to a certain size and divide
into two daughter cells.
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Sexual reproduction
This involves the formation of a zygote (a diploid cell) from the fusion of two haploid
cells, each having a single set of chromosomes. e.g. some yeasts can exist as haploid (in
the forms of  and a cells) or diploid (formed by mating of  and a cells). The haploid
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Bud scars are observable under microscope. One cell can undergo multiple divisions # of bud scars can be used to assess
cellular age because a scar represents a complete cell division.
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contains 16 linear chromosomes each consisting of 3 essential regions for replication:
ARS (autonomous replication sequence), centromeres and telomeres.

Yeast DNA is located within the nucleus and the modification of mRNA (5’ G-cap and
3’ poly A) is similar to that of higher eukaryotes.
Molds (filamentous fungi):

Have a mycelial (菌絲) 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).
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When grown in submerged culture, molds often form cell aggregates and pellets. Pellet
formation can cause nutrient transfer problems. However, pellet formation reduces broth
viscosity, which can improve bulk oxygen transfer.
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Molds are used for the production of citric acid (e.g. Aspergillus niger) and many
antibiotics (e.g. Penicillium chrysogenum).
when a conidia spore (無性
孢子) lands on a suitable
substrate, it germinates and
develops into hyphae
II. Introducing DNA into fungi (fungi transformation)
General procedures (for filamentous fungi):

Prepare the recombinant DNA as in Chap 4.
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Grow the cells, and remove the cell walls by incubating the cells in a buffer
containing the carbohydrase and osmotic stabilizer (to prevent cells from
bursting).
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
Wash protoplasts2 with buffer containing the osmotic stabilizer.
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Add plasmid DNA, CaCl2 and polyethylene glycol (PEG induces the uptake of
DNA) to the cells.
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Select the colonies that contain the foreign genes.
This protocol also applies to some yeasts such as S. cerevisiae because S. cerevisiae
also produces spores. However, yeast can be commonly transformed with lithium
acetate (just like E. coli transformation), which can provide a high transformation
efficiency of 105 to 106 transformants per g DNA.
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Various protocols (e.g. electroporation) have been devised to enhance the
transformation efficiency, but these also suffer from the limitations of suitable host
range and the need for specialized equipments.
Vectors
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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).
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fungal cell lack of cell wall
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
Shuttle vector: plasmid that can
Features of shuttle vector:
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Three groups of selectable markers:
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Genes with antibiotics resistance, e.g. hygromycin, kanamycin, etc.
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Genes that can complement3 auxotrophic4 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.
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Genes that confer the ability to grow on C or N sources which the host strain would
not normally be able to use.
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Genetic complementation: the phenomenon that
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Auxotrophic mutant: a mutant strain requiring a specific nutrient (e.g. amino acid or dNTP or NTP) to survive.
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
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.
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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.
Integration into chromosomes

Plasmid can survive in the yeast but typically foreign genes must be integrated into the
filamentous fungi.
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Leads to enhanced stability, but lower number of introduced gene
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May not carry ori in the shuttle vector so that only cells w/ foreign genes integrated can
survive in the presence of selective pressure.
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Can be achieved by
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.
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The gene copy number is lower. 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.
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The integration site influences the subsequent expression level.
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III.Biological applications of fungi
e.g. S. cerevisiae (baker’s yeast) contains abundant proteins, vitamin D and B, and Ca, Fe, Zn, K, P, Na (trace elements) a good single
cell protein source (SCP).
The importance of secretion on protein production

Most commercial enzymes are secreted from the source cells. Secreted enzymes are
usually correctly folded and active because this is a function of the secretory pathway.
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Overproduction of intracellular proteins can lead to the accumulation of improperly
folded and inactive proteins. Also, the extraction process may inactivate a proportion of
the protein, thus reducing recoverable yields.

So, high secretion efficiency is desired==> those species that naturally secrete enzymes
as part of lifestyles might be the systems of choice. In particular, filamentous fungi
secrete enzymes to degrade polymeric matters surrounding them, so filamentous fungi
are commonly used for commercial enzyme production.
Yeasts for heterologous proteins production

S. cerevisiae
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A yeast used in the production of bread and alcohol, is regarded as safe, and its gene
transfer and gene regulation/expression have been extensively studied.
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Widely used for protein production (e.g. human insulin, HBsAg (hepatitis B surface
antigen), HPV VLP (human papilloma virus-like particle, Gardasil from Merck)).
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Problem: Hyperglycosylation: N-linked (linked to arginine) carbohydrates are often
extremely long and of high-mannose type which is not characteristic of human
glycans.
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Alternatives:
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K. lactis: can be grown on lactose-containing whey (乳清5); has strong, inducible
promoters to drive the expression; has been used for the commercial production of
chymosin.
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Pichia angusta and Pichia. pastoris: methanol utilizing yeasts; posses strong,
methanol-inducible promoter from methanol oxidase gene. Secrection in both
species are high and hyperglycosylation appears not to be a problem.
Heterologous proteins from filamentous fungi
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The features of the expression vectors are similar to those of yeast. The only difference
is, because autonomous plasmid replication is not normally an option in commercial
filamentous fungi, most vectors are designed to integrate into the fungal genome.
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Multiple copies of genes can be introduced but there is a limit in the gene numbers
because essential cellular resources (e.g. transcription factors) may become limiting. The
limitation may be overcome by up-regulating the expression of the limiting factor (a part
of metabolic engineering).
References:
1.
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Shuler ML and Kargi F. (1992) Bioprocess Engineering: Basic Concepts. Prentice Hall
Whey is the liquid remaining after milk has been curdled (凝固) and strained. It is a by-product of the
manufacture of cheese or casein (酪蛋白).
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2.
IV.
International, London.
Ausubel, FM, Brent, R, Kingston, RE, Moore, DD, Seidman, JG, Smith, JA, Struhl, K.
(1999) Short protocols in molecular biology. 4th Ed. John Wiley & Sons, New York.
Appendix
Gene Isolation by PCR
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PCR is now frequently used to isolate the genes.
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Requires the information of the gene sequences to be cloned (from a known gene) for
the design of primers (which encode the highly conserved region).
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For gene cloning from an unknown gene, the protein (the gene product) sequence needs
to be identified. Because the genetic code is redundant, i.e. more than one codon can
encode the same amino acids, the primers are usually mixtures of different DNA which
nevertheless encode the same amino acid sequence. This approach would generate
many different PCR fragment species and gives a smeared appearance after
electrophoresis.
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A second round of PCR with “nested primers” (a second set of primers which are
internal to the first set, and designed from additional conserved regions) can help to
alleviate this problem.
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