Chap 5 Genetic Engineering: yeast and filamentous fungi

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Dec 11, 2012 (4 years and 4 months ago)

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1

Chap 5 Genetic Engineering: yeast and filamentous fungi

I.

Introduction



Fungi range in size from microscopic to macroscopic (e.g. mushroom) forms.
Microscopic f
ungi
include

yeasts (usually unicellular) and filamentous (
細絲狀
) fungi
(e.g. molds
黴菌
)
.




Fungal ce
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
(e.g.
S. cerevisiae
: 16 pairs;
Aspergillus nidulans

(
麴菌
)
: 8).



Fungi contain a larger genome (>10 Mb compared to 4.7 MB for
E. coli
)
because

fungi
have more genes, and more DNA which does not code for proteins
.

These noncoding
DNA are located
within genes as introns or as spacer DNA between genes.



Genes in fung
i are
monocistronic

(polycistronic in bacteria)
.

Yeasts:
(See Shuler Chap. 2 and Ausubel Chap 13)



S
ingle cells of
typically
5
-
10

m

(but can vary from 2
-
3

m to 20
-
50

m)
. Either
spherical, cylindrical or oval.


2



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
a
sexual or sexual means.



Asexual

reproduction
:



budding
:

a small bud
1

forms on the cell, which gradually enlarges
and separates
from the mother cell.



Fission
:

similar to that of bacteria. In fission, cells grow to a certain size and divide
into two equal cells.




Sexual reproduction

This
involves the formation of a zygote (a
diploid

cell) from
the
fusion of two ha
ploid
cells, each having a single set of chromosomes.

e.g.
Some
yeast
s

can exist in haploid (in
the forms of


a湤n
a

cells) or diploid (formed by mating of


a湤n
a

cells). The haploid
contains 16 linear chromosomes
each consisting of
3 essential regions f
or replication:
ARS (autonomous replication

sequence), centromeres and telo
meres.




1

Bu
d scars are observable under microscope. One cell can undergo multiple divisions

# of bud scars can be used to assess cellular age
because

a scar
represent
s a complete cell division.


3



Yeast DNA is located within the nucleus and the modifications of mRNA (5


G
-
cap and
3


poly A) is similar to that of higher
eukaryotes
.



The yeast can grow mitotically indefin
itely
,

but under conditions of C and N starvation


meiosis


production of spores.

Molds:



Filamentous fungi that have a mycelial (
菌絲
) structure which is
a
highly branched
system of tubes that contains mobil
e

cytoplasm with many nuclei.
A
single
l
ong thin
filament on the
mycelium

is called a hypha (plural:

hyphae
).




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 (
Aspergillus niger
) and many antibiotics

(
Penicillium chrysogenum
).



II.

Introducing DNA into fungi (fungi transformation)

Terminologies



2
Auxotrophi
c mutant:

a mutant strain requiring an amino acid or
dNTP or NTP
to
survive.



Protoplast:

fungal cell
lack of

cell wall
(can be
removed by carbohydrase
)
.




2

auxo
-
=increase, as an increase in requirements

when a conidia

spore
(
無性
孢子
)

lands on a suitable
substrate, it germinates and
develops into hyphae


4



Genetic complementation:

the

phenomenon

that
a gene convert
s

a mutant phenotype to
wild
-
type

General pr
ocedures

(for filamentous fungi)
:




Prepare the recombinant
DNA
as in Chap 4 for
pro
c
aryotic

cells
.



Grow the cells, and r
emove the cell walls by incubating the cells
in a buffer
containing
the carbohydrase and
osmotic stabilizer
(
to prevent cells from
burs
ting
)
.



Add plasmid DNA, CaCl
2

and polyethylene glycol (PEG induces the uptake of
DNA).



Select

the colonies that contain the foreign genes
.




This protocol also applies to yeast
s

such as

S. cerevisiae

because
S. cerevisiae

also
produces spores (efficiency i
s higher). However, yeast can be commonly transformed
with lithium acetate (just like
E. coli

transformation

which
appl
ies

chemicals and a heat
shock) which can provide a high transformation efficiency of 10
5

to 10
6

transformants
per

g DNA.


5



Various prot
ocols have been devised to enhance the transformation efficiency (e.g.
electroporation)
,

but these also suffer from the limitations of suitable host range and
the need for specialized equipment
s
.

V
ectors



Can be designed to introduce DNA which either integ
rates into the genomic DNA
(for
most filamentous fungi)
or can be maintained as a plasmid

(for some yeasts)
.



Shuttle vector
: specially
-
designed plasmid

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
s

the experimental work.




Features of shuttle vector:



C
ontain
s

bacterial and yeast origins of replication (
many carry ARS from 2

m circle
which is a naturally occurring plasmid in most lab strains of
S. cerevisiae
.
T
he
ori

typically allows the maintenance of 10
-
40
copies
per cell
.



Select
able
markers for
E. coli

and yeasts



promoter, terminator and MCS



Three groups of select
able

markers:

1.

Genes with antibiotics resistance, such as hygromycin, kanamycin, etc.

2.

Genes that can
complement auxotrophic growth requirements
. M
any of the
yeast markers encode functions that are involved in biosynthesis pathways of

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yeast,
e.g. URA3 gene essential for uracil synthesis can complement
ura3
-

mutants so these vectors must be transformed into

the auxotrophic mutants
.

3.

Genes that confer the ability to grow on
C

or
N

sources which the host strain
would not normally be able to use.



Plasmid vectors are maintained provided
that
the transformants are grown under
selective
pressure
. Once the selectiv
e 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 20
0
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.



Leads to e
nhanced stability but lower number of intr
oduced gene



May not carry
o
ri

in the shuttle vector
so that only cells w/ foreign genes integrated can
survive in the presence of selective pressure.



Can be achieved by
homologous
recombination
:
exchange of DNA
between the vector DNA and the
genomic DNA d
ue to the similarity in
DNA sequences.



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



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 repeat
s

per genome.



The
integration site influences the subsequent expression level.



7

III.

Biological applications of fungi


e.g.
S. cerevisiae

(baker

s yeast
)

con
tains abundant proteins, vitamin D and B, and Ca, Fe, Zn, K, P, Na (trace elements)


a good single cell
protein source (SCP).

T
he importance of secretion

on protein production



Most commercial enzymes are secreted from the source cells. Secreted enzymes ar
e
usually correctly folded and active
because

this is a function of the secretory pathway.



Overproduction of intracellular proteins can lead to the accumulation of improperly
folded and inactive protein.
Also,
the extraction process may inactivate a prop
ortion 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 deg
rade polymeric matters
surrounding them,
so filamentous fungi
are commonly used for
commercial

enzyme
production
.

Yeasts for h
eterologous proteins
production



S. cerevisiae


8



a yeast used in the production of bread
and

alcohol
,

is regarded as safe
,

and
its
g
ene
transfer and gene regulation/expression have been extensively studied.



Widely used for protein production (e.g.
human insulin,
HBsAg
, HPV VLP
(Gardasil


from Merck)
).



Problem:

Hyperglycosylation: N
-
linked (linked to arginine) carbohydrates are often
e
xtremely long and of high
-
mannose type which is not characteristic of human
glycans
.



Alternatives:



K. lactis
:
grown

on lactose
-
containing whey

(
乳清
)
, has strong, inducible promoter
s

to drive the expression. It has been used for the
commercial

production of

chymosin.



Pichia angusta

and
P. pastoris
:

methanol utilizing yeast, posses strong, methanol
-
inducible promoter fr
o
m methanol oxidase gene. Secrection in both species are
high and hyperglycosylation appear
s

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
is,
because

autonomous replication is not normally an option i
n

commercial

filamentous

fungi, most vectors are designed to integrate into the fun
gal genome.



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 li
miting factor (a part
of metabolic engineering).


References:

1.

Shuler ML and Kargi F. (1992) Bioprocess Engineering: Basic Concepts. Prentice Hall
International, London.

2.

Ausubel, FM, Brent, R, Kingston, RE, Moore, DD, Seidman, JG, Smith, JA, Struhl, K.
(19
99) Short protocols in molecular biology. 4
th

Ed. John Wiley & Sons, New York.




9

IV.

Appendix

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
the design

of
primer
s (
which encode the highly conserved region
)
.



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, 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
electrophoresis.



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.