5 research outputs found
Synthetic Core Promoters for <i>Pichia pastoris</i>
Synthetic
promoters are commonly used tools for circuit design
or high level protein production. Promoter engineering efforts in
yeasts, such as <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i> have mostly been focused on altering upstream regulatory
sequences such as transcription factor binding sites. In higher eukaryotes
synthetic core promoters, directly needed for transcription initiation
by RNA Polymerase II, have been successfully designed. Here we report
the first synthetic yeast core promoter for <i>P. pastoris</i>, based on natural yeast core promoters. Furthermore we used this
synthetic core promoter sequence to engineer the core promoter of
the natural <i>AOX1</i> promoter, thereby creating a set
of core promoters providing a range of different expression levels.
As opposed to engineering strategies of the significantly longer entire
promoter, such short core promoters can directly be added on a PCR
primer facilitating library generation and are sufficient to obtain
variable expression yields
Synthetic Core Promoters for <i>Pichia pastoris</i>
Synthetic
promoters are commonly used tools for circuit design
or high level protein production. Promoter engineering efforts in
yeasts, such as <i>Saccharomyces cerevisiae</i> and <i>Pichia pastoris</i> have mostly been focused on altering upstream regulatory
sequences such as transcription factor binding sites. In higher eukaryotes
synthetic core promoters, directly needed for transcription initiation
by RNA Polymerase II, have been successfully designed. Here we report
the first synthetic yeast core promoter for <i>P. pastoris</i>, based on natural yeast core promoters. Furthermore we used this
synthetic core promoter sequence to engineer the core promoter of
the natural <i>AOX1</i> promoter, thereby creating a set
of core promoters providing a range of different expression levels.
As opposed to engineering strategies of the significantly longer entire
promoter, such short core promoters can directly be added on a PCR
primer facilitating library generation and are sufficient to obtain
variable expression yields
A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in <i>Pichia pastoris</i>
The heterologous expression of biosynthetic
pathways for pharmaceutical
or fine chemical production requires suitable expression hosts and
vectors. In eukaryotes, the pathway flux is typically balanced by
stoichiometric fine-tuning of reaction steps by varying the transcript
levels of the genes involved. Regulated (inducible) promoters are
desirable to allow a separation of pathway expression from cell growth.
Ideally, the promoter sequences used should not be identical to avoid
loss by recombination. The methylotrophic yeast Pichia
pastoris is a commonly used protein production host,
and single genes have been expressed at high levels using the methanol-inducible,
strong, and tightly regulated promoter of the <i>alcohol oxidase
1</i> gene (<i>P</i><sub><i>AOX1</i></sub>). Here, we have studied the regulation of the P.
pastoris methanol utilization (MUT) pathway to identify
a useful set of promoters that (i) allow high coexpression and (ii)
differ in DNA sequence to increase genetic stability. We noticed a
pronounced involvement of the pentose phosphate pathway (PPP) and
genes involved in the defense of reactive oxygen species (ROS), providing
strong promoters that, in part, even outperform <i>P</i><sub><i>AOX1</i></sub> and offer novel regulatory profiles.
We have applied these tightly regulated promoters together with novel
terminators as useful tools for the expression of a heterologous biosynthetic
pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest
sets of strong and co-regulated promoters of any microbe, moving it
from a protein production host to a general industrial biotechnology
host
A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in <i>Pichia pastoris</i>
The heterologous expression of biosynthetic
pathways for pharmaceutical
or fine chemical production requires suitable expression hosts and
vectors. In eukaryotes, the pathway flux is typically balanced by
stoichiometric fine-tuning of reaction steps by varying the transcript
levels of the genes involved. Regulated (inducible) promoters are
desirable to allow a separation of pathway expression from cell growth.
Ideally, the promoter sequences used should not be identical to avoid
loss by recombination. The methylotrophic yeast Pichia
pastoris is a commonly used protein production host,
and single genes have been expressed at high levels using the methanol-inducible,
strong, and tightly regulated promoter of the <i>alcohol oxidase
1</i> gene (<i>P</i><sub><i>AOX1</i></sub>). Here, we have studied the regulation of the P.
pastoris methanol utilization (MUT) pathway to identify
a useful set of promoters that (i) allow high coexpression and (ii)
differ in DNA sequence to increase genetic stability. We noticed a
pronounced involvement of the pentose phosphate pathway (PPP) and
genes involved in the defense of reactive oxygen species (ROS), providing
strong promoters that, in part, even outperform <i>P</i><sub><i>AOX1</i></sub> and offer novel regulatory profiles.
We have applied these tightly regulated promoters together with novel
terminators as useful tools for the expression of a heterologous biosynthetic
pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest
sets of strong and co-regulated promoters of any microbe, moving it
from a protein production host to a general industrial biotechnology
host
A Toolbox of Diverse Promoters Related to Methanol Utilization: Functionally Verified Parts for Heterologous Pathway Expression in <i>Pichia pastoris</i>
The heterologous expression of biosynthetic
pathways for pharmaceutical
or fine chemical production requires suitable expression hosts and
vectors. In eukaryotes, the pathway flux is typically balanced by
stoichiometric fine-tuning of reaction steps by varying the transcript
levels of the genes involved. Regulated (inducible) promoters are
desirable to allow a separation of pathway expression from cell growth.
Ideally, the promoter sequences used should not be identical to avoid
loss by recombination. The methylotrophic yeast Pichia
pastoris is a commonly used protein production host,
and single genes have been expressed at high levels using the methanol-inducible,
strong, and tightly regulated promoter of the <i>alcohol oxidase
1</i> gene (<i>P</i><sub><i>AOX1</i></sub>). Here, we have studied the regulation of the P.
pastoris methanol utilization (MUT) pathway to identify
a useful set of promoters that (i) allow high coexpression and (ii)
differ in DNA sequence to increase genetic stability. We noticed a
pronounced involvement of the pentose phosphate pathway (PPP) and
genes involved in the defense of reactive oxygen species (ROS), providing
strong promoters that, in part, even outperform <i>P</i><sub><i>AOX1</i></sub> and offer novel regulatory profiles.
We have applied these tightly regulated promoters together with novel
terminators as useful tools for the expression of a heterologous biosynthetic
pathway. With the synthetic biology toolbox presented here, P. pastoris is now equipped with one of the largest
sets of strong and co-regulated promoters of any microbe, moving it
from a protein production host to a general industrial biotechnology
host