Chap 5 Genetic Engineering: yeast and filamentous fungi
Fungi range in size from microscopic to macroscopic (e.g. mushroom) forms.
yeasts (usually unicellular) and filamentous (
ll walls do not contain peptidoglycan which is found only in bacteria. Rather,
their walls are composed primarily of
polysaccharides (glucans, chitin, chitosan).
Eucaryotes with multiple chromosomes
: 16 pairs;
Fungi contain a larger genome (>10 Mb compared to 4.7 MB for
have more genes, and more DNA which does not code for proteins
DNA are located
within genes as introns or as spacer DNA between genes.
Genes in fung
(polycistronic in bacteria)
(See Shuler Chap. 2 and Ausubel Chap 13)
ingle cells of
(but can vary from 2
m to 20
spherical, cylindrical or oval.
Can grow well on a minimal medium containing
dextrose as a C source and salts that
supply N, P and trace metals. Under optimal growth conditions, doubling time=90 min.
Can reproduce by
sexual or sexual means.
a small bud
forms on the cell, which gradually enlarges
from the mother cell.
similar to that of bacteria. In fission, cells grow to a certain size and divide
into two equal cells.
involves the formation of a zygote (a
fusion of two ha
cells, each having a single set of chromosomes.
can exist in haploid (in
the forms of
cells) or diploid (formed by mating of
cells). The haploid
contains 16 linear chromosomes
each consisting of
3 essential regions f
ARS (autonomous replication
sequence), centromeres and telo
d scars are observable under microscope. One cell can undergo multiple divisions
# of bud scars can be used to assess cellular age
s a complete cell division.
Yeast DNA is located within the nucleus and the modifications of mRNA (5
poly A) is similar to that of higher
The yeast can grow mitotically indefin
but under conditions of C and N starvation
production of spores.
Filamentous fungi that have a mycelial (
) structure which is
system of tubes that contains mobil
cytoplasm with many nuclei.
filament on the
is called a hypha (plural:
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 c
an improve bulk oxygen transfer.
Molds are used for the production of citric acid (
) and many antibiotics
Introducing DNA into fungi (fungi transformation)
a mutant strain requiring an amino acid or
dNTP or NTP
removed by carbohydrase
=increase, as an increase in requirements
when a conidia
lands on a suitable
substrate, it germinates and
develops into hyphae
a gene convert
a mutant phenotype to
(for filamentous fungi)
Prepare the recombinant
as in Chap 4 for
Grow the cells, and r
emove the cell walls by incubating the cells
in a buffer
the carbohydrase and
to prevent cells from
Add plasmid DNA, CaCl
and polyethylene glycol (PEG induces the uptake of
the colonies that contain the foreign genes
This protocol also applies to yeast
produces spores (efficiency i
s higher). However, yeast can be commonly transformed
with lithium acetate (just like
chemicals and a heat
shock) which can provide a high transformation efficiency of 10
ocols have been devised to enhance the transformation efficiency (e.g.
but these also suffer from the limitations of suitable host range and
the need for specialized equipment
Can be designed to introduce DNA which either integ
rates into the genomic DNA
most filamentous fungi)
or can be maintained as a plasmid
(for some yeasts)
that can be propagated in E.
coli and yeast (or
other host cells). This design enables the genetic mani
pulations in E.
coli and facilitate
the experimental work.
Features of shuttle vector:
bacterial and yeast origins of replication (
many carry ARS from 2
which is a naturally occurring plasmid in most lab strains of
typically allows the maintenance of 10
promoter, terminator and MCS
Three groups of select
Genes with antibiotics resistance, such as hygromycin, kanamycin, etc.
Genes that can
complement auxotrophic growth requirements
any of the
yeast markers encode functions that are involved in biosynthesis pathways of
e.g. URA3 gene essential for uracil synthesis can complement
mutants so these vectors must be transformed into
the auxotrophic mutants
Genes that confer the ability to grow on
sources which the host strain
would not normally be able to use.
Plasmid vectors are maintained provided
the transformants are grown under
. Once the selectiv
, the plasmids could be lost during the
Plasmid vectors can replicate with
from one yeast strain can normally function
in different yeast hosts, albeit not always with the same degree of efficiency. Up to 20
copies can be present in a single cell
Integration into chromosomes
Plasmid can survive in the yeast but typically foreign genes must be integrated into the
Leads to e
nhanced stability but lower number of intr
May not carry
in the shuttle vector
so that only cells w/ foreign genes integrated can
survive in the presence of selective pressure.
Can be achieved by
exchange of DNA
between the vector DNA and the
genomic DNA d
ue to the similarity in
Integration can also be used to disrupt
or replace a desired gene,
which can be
o test the function of each
gene in the cell.
The gene copy number is lower.
the number of genes in
is to integrate into
which can be present at about 150 tandem repeat
integration site influences the subsequent expression level.
Biological applications of fungi
tains abundant proteins, vitamin D and B, and Ca, Fe, Zn, K, P, Na (trace elements)
a good single cell
protein source (SCP).
he importance of secretion
on protein production
Most commercial enzymes are secreted from the source cells. Secreted enzymes ar
usually correctly folded and active
this is a function of the secretory pathway.
Overproduction of intracellular proteins can lead to the accumulation of improperly
folded and inactive protein.
the extraction process may inactivate a prop
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 deg
rade polymeric matters
so filamentous fungi
are commonly used for
Yeasts for h
a yeast used in the production of bread
is regarded as safe
transfer and gene regulation/expression have been extensively studied.
Widely used for protein production (e.g.
, HPV VLP
linked (linked to arginine) carbohydrates are often
xtremely long and of high
mannose type which is not characteristic of human
, has strong, inducible promoter
to drive the expression. It has been used for the
methanol utilizing yeast, posses strong, methanol
inducible promoter fr
m methanol oxidase gene. Secrection in both species are
high and hyperglycosylation appear
not to be a problem.
Heterologous proteins fro
m filamentous fungi
The features of the expression vectors are similar to those of yeast. The only difference
autonomous replication is not normally an option i
fungi, most vectors are designed to integrate into the fun
Multiple copies of genes can be introduced but there is a limit in the gene numbers
essential cellular resources (e.g. transcription factors) may become limiting. The
limitation may be overcome by up
regulating the expression of the li
miting factor (a part
of metabolic engineering).
Shuler ML and Kargi F. (1992) Bioprocess Engineering: Basic Concepts. Prentice Hall
Ausubel, FM, Brent, R, Kingston, RE, Moore, DD, Seidman, JG, Smith, JA, Struhl, K.
99) Short protocols in molecular biology. 4
Ed. John Wiley & Sons, New York.
Gene Isolation by PCR
PCR is now frequently used to isolate the genes.
Requires the information of the gene sequences to be cloned
(from a known gene) for
which encode the highly conserved region
For gene cloning from an unknown gene,
(the gene product) sequence needs
to be identified
the genetic code is redundant, i.e. more than one codon can
encode the same amino acids, t
he primers are usually
mixtures of different DNA which
nevertheless encode the same amino acid sequence. This approach would generate
many different PCR fragment sp
ecies and gives a smeared appearance after
A second round of PCR with
(a second set of primers which are
internal to the first set, and designed from additional conserved regions) can help to
alleviate this problem.