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Notes

Uploaded by

habino
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© © All Rights Reserved
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Synechocystis does not naturally produce fumarate.

However, model guided engineering found that


removing a single gene within Synechocystis leads to a stable cell factory that produces fumarate as it
grows during the day. Nevertheless, at night our cells do not produce fumarate, since at night, they
don't grow. To overcome this challenge, we have taken a systems biology approach which interweaves
theory, modeling, and experimentation to implement stable nighttime production of fumarate. We
theorized that we can redirect the nighttime flux towards fumarate production by removing a competing
pathway via knockout of the zwf gene. Additionally, we also took inspiration from nature and speculated
that the incorporation of the glyoxylate shunt would further increase our nighttime production of
fumarate. Our models corroborate these predictions, however, they also suggest that the stability of the
glyoxylate shunt is sensitive to the timing of when the shunt is turned on (i.e. expressed). We therefore
took a robust approach to incorporate the glyoxylate shunt enzymes under ideal expression conditions.
Highlights Engineered a ΔfumCΔzwf Synechocystis strain, that uses different fumarate production
strategies during day and night. Developed a method to make fully segregated libraries in polyploid
organisms Created the first fully segregated library representing the entire genome (99.9% confidence)
of Synechocystis upstream of the glyoxylate shunt genes. This library is now ready to be tested to
further increase nighttime fumarate production. Stable production of fumarate directly from CO around
the clock Qp of 18.4 μM grDW hour Qp of 58.77 μM grDW hour ASynechocystis does not naturally
produce fumarate. However, model guided engineering found that removing a single gene within
Synechocystis leads to a stable cell factory that produces fumarate as it grows during the day.
Nevertheless, at night our cells do not produce fumarate, since at night, they don't grow. To overcome
this challenge, we have taken a systems biology approach which interweaves theory, modeling, and
experimentation to implement stable nighttime production of fumarate. We theorized that we can
redirect the nighttime flux towards fumarate production by removing a competing pathway via knockout
of the zwf gene. Additionally, we also took inspiration from nature and speculated that the
incorporation of the glyoxylate shunt would further increase our nighttime production of fumarate. Our
models corroborate these predictions, however, they also suggest that the stability of the glyoxylate
shunt is sensitive to the timing of when the shunt is turned on (i.e. expressed). We therefore took a
robust approach to incorporate the glyoxylate shunt enzymes under ideal expression conditions.
Highlights Engineered a ΔfumCΔzwf Synechocystis strain, that uses different fumarate production
strategies during day and night. Developed a method to make fully segregated libraries in polyploid
organisms Created the first fully segregated library representing the entire genome (99.9% confidence)
of Synechocystis upstream of the glyoxylate shunt genes. This library is now ready to be tested to
further increase nighttime fumarate production. Stable production of fumarate directly from CO around
the clock Qp of 18.4 μM grDW hour Qp of 58.77 μM grDW hour ASynechocystis does not naturally
produce fumarate. However, model guided engineering found that removing a single gene within
Synechocystis leads to a stable cell factory that produces fumarate as it grows during the day.
Nevertheless, at night our cells do not produce fumarate, since at night, they don't grow. To overcome
this challenge, we have taken a systems biology approach which interweaves theory, modeling, and
experimentation to implement stable nighttime production of fumarate. We theorized that we can
redirect the nighttime flux towards fumarate production by removing a competing pathway via knockout
of the zwf gene. Additionally, we also took inspiration from nature and speculated that the
incorporation of the glyoxylate shunt would further increase our nighttime production of fumarate. Our
models corroborate these predictions, however, they also suggest that the stability of the glyoxylate
shunt is sensitive to the timing of when the shunt is turned on (i.e. expressed). We therefore took a
robust approach to incorporate the glyoxylate shunt enzymes under ideal expression conditions.
Highlights Engineered a ΔfumCΔzwf Synechocystis strain, that uses different fumarate production
strategies during day and night. Developed a method to make fully segregated libraries in polyploid
organisms Created the first fully segregated library representing the entire genome (99.9% confidence)
of Synechocystis upstream of the glyoxylate shunt genes. This library is now ready to be tested to
further increase nighttime fumarate production. Stable production of fumarate directly from CO around
the clock Qp of 18.4 μM grDW hour Qp of 58.77 μM grDW hour A

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