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Genetic improvement
of recombinant protein
production in
Kluyveromyces lactis.
Why Kluyveromyces lactis for biotechnology?
It is lactose-positive, able to grow
on poor substrates such as whey
-
It is a safe organism
for
industrial use (GRAS)
characteristics
shared
by regulated
S. cerevisiae
Strong and
promoters and
engineered vectors are available
It has good secretory properties
It is capable of secreting
large proteins into the medium
To identify the best genetics and physiological
conditions for the production of the
heterologous protein we choose as model
system
the glucoamylase
from Arxula adeninivorans
Secretion efficiency can be directly detected
on starch plates as clear halo by the digestion
of starch around the cell
Ampr
URA3
Sal I
P-GAP
Hind III
Cla I
GAA
pTS32x-GAA
Sma I
B
(11.69 kb)
EcoR I
Sal I
EcoR I
T-PHO5
C
Hind III
Sma I
Bui et al.,
A
Improvement of heterologous protein
production
Choice of the strain
(secretion phenotype)
Control of
host physiology
(carbon source)
starch
the level of glucoamylase produced and
PM4-4B
secreted into the medium is
JA6
strain dependent
2359
MW270 and carbon source dependent
Starch + lactose
Starch + glucose
starch
PM4-4B
JA6
2359
MW270
Starch + lactose
Starch + glucose
JA6 is the best producer
starch
PM4-4B
JA6
2359
MW270
Starch + lactose
Starch + glucose
Lactose is the best condition and glucose the worst
starch
PM4-4B
JA6
2359
MW270
Starch + lactose
Starch + glucose
The reduced halo dimension on glucose respect to that observed on
starch suggest that glucoamylase productionwas glucose-repressed
Glucose
0,2%
1%
5%
Effect
of glucose concentration
the level
of heterologous
protein produced
on gluco-amylase
production
and secreted
into the medium
depends on glucose concentration
We have therefore analysed the level of secreted
glucoamylase in the non repressible mutants
dgr151, rag1kht2 and mig1.
Gene
Coded protein
Mutant phenotype
RAG1*
Coded protein
Coded protein
DGR151
Coded protein
Coded protein
MIG1
Coded protein
Coded protein
* Il locus RAG1 è polimorfico. In JA6 un secondo gene, KHT2, è arrangiato in tandem a RAG1.
starch
rag1hxt1
dgr151
starch
+
lactose
wild type
mig1
starch
+
glucose
rag1hxt1 and dgr151 mutation positively affects production
in glucose confirming the glucose repression of production.
starch
rag1hxt1
dgr151
starch
+
lactose
wild type
mig1
starch
+
glucose
dgr151show a better production also in starch and lactose
suggesting a role of DGR151 independent of its role in
glucose repression
starch
rag1hxt1
dgr151
starch
+
lactose
wild type
mig1
starch
+
glucose
mig1 is severely affected in all the conditions suggesting a
positive and consistent role of MIG1
dgr151 is able to iperproduce also other
secreted recombinant proteins?
To answer to this question we analysed the levels of :
- Human serum albumin (HSA)
- Interleukin 1b (IL-1b)
Protein: Human interleukin-1b
Cultural condition: YEPD deprived of phosphate
Promoter: PHO5
HIL-1b
dgr151 show an increased production of H-interleuchina1b
Protein: Human serum albumin
Cultural condition: YEPD + ethanol 1%
Promoter: ADH4
HSA
dgr151 show an increased production of
Human serum albumin
Cell Wall analysis
dgr151
JA6
dgr151 do not show morphological defects
Cell Wall functionality
Saggio Zymoliasi 28° C
Saggio Zimoliasi 37°C
120
JA6
100
100
80
JA6
80
% cellule vitali
% cellule vitali
90
dgr
60
40
70
dgr
60
50
40
30
20
20
10
0
0
0
10
20
30
Tempo (min)
40
50
60
0
5
10
15
20
25
30
35
40
Tempo (min)
dgr151 do not show functional defects
45
50
55
Analysis of dgr151 mutation
•Comparison of wild-type and dgr151 protein
G176D
WT
dgr151
R206A
Istituto di Genetica,
Università di Parma
Barbara Neglia
Paola Goffrini
Iliana Ferrero
Claudia Donnini
Dipartimento di Biologia
Cellulare e dello Sviluppo
Università di Roma
"La Sapienza"
Francesca Farina
Francesca La Starza
Claudio Palleschi
Acknowledgements
MINISTERO DELL’UNIVERSITA’ E DELLA
RICERCA SCIENTIFICA
- Scientific Research Pogramms of rilevant national interest
COFIN 2000
Biotechnology of yeasts: optimisation of the production
and secretion of heterologus proteins.
Coordinator:Prof. Danilo Porro