Team:DTU Denmark/applications private securkey Dhjg1mab2ak47

There are numerous potential applications for our genetic device, but here we have highlighted three key applications: an in vivo redox sensor, improved chemostat product formation and applications in cancer research.

The gene encoding green fluorescent protein (GFP) is widely used as a reporter gene in molecular biology. By placing the ROB promoter upstream of a GFP gene on a plasmid, and transforming the whole system into a yeast cell, GFP will be expressed at certain NAD⁺/NADH levels. When the REX-activator protein is bound to DNA, GFP expression will produce a visible and quantitatively measurable signal, which will be an indirect measure of the NAD⁺/NADH ratio.

In vivo online monitoring of the redox poise

A Redoxilator plasmid can be transformed into any yeast cell to analyse the NAD⁺/NADH level by online measurement of reporter gene expression. The internal redox level of the cell is very indicative and have serious impact on product formation of many products

The measuring of cellular NAD⁺/NADH levels is usually a difficult process, especially due to fast changes in NAD⁺/NADH ratio that can occur when a sample is taken and exposed to slightly new conditions. With this sensor system, the quantitative measurement of the reporter gene will provide a fast and reliable way to determine the NAD⁺/NADH ratio in vivo. The plasmid can be transformed into a yeast strain allowing the NAD⁺/NADH ratio to be continuously monitored. As an example this plasmid can be used to study whether an engineered yeast strain has an altered NAD⁺/NADH ratio. The plasmid can be transformed into different yeast strains (e.g. wild type versus an engineered strain or two production strains) and the NAD⁺/NADH ratio can be compared.

When S. cerevisiae are grown continuously in a chemostat, the productivity of e.g. antibiotic or protein product gradually decreases [Personal correspondence with Novo Nordisk A/S]. This occurs during maximum production and is believed to be the result of metabolic adaption with reduced product formation as a consequence. The metabolic adaption is believed to occur because the cells are stressed by the extensive production. The cells will adapt to the new metabolic situation, which will gradually lead to lower production rates. This is highly undesirable in the biotech industry, as the chemostats will have to be restarted on a regular basis, which is costly and time consuming.

Qualitative simulation of a chemostat fermentation with the Redoxilator Production system

Chemostat oscillations in the NAD+/NADH ratio as a result of the Yeast Metabolic Cycle (A: top graph) lead to change in the Rex DNA binding affinity (B: middle graph) leading to controlled bursts in product of reporter gene expression (C: lower graph).

A strategy to lower the effect of the metabolic adaption could be to couple the productivity with the yeast metabolic cycle (see grey box to the right). The NAD⁺/NADH poise oscillates in parallel with the yeast metabolic cycle. The synthesis of a given product will be put under control of the Redoxilator, which will lead to periodic burst of gene expression leading to product formation. Consequently the cells will have time to recover periodically from the metabolic stress that occurs during the production phase. There are many indications that this will lead to a prolonged effective chemostat operation, and ultimately higher accumulation of product before a costly restart of the chemostat is required.

The results of several fermentations gives a strong indication that it is favourable to let the cells produce in the oxidative phase for the tested product (the specific product cannot be mentioned due to ip-rights) (Bonde, 2009).

iii) Yeast as a model organism for humans: Redoxilator in cancer research

Yeast is used as a model organism for humans in many research projects. As an example, studies of the yeast cell cycle have paved the way for most of the knowledge about the cell cycle in mammalian cells. As yeast has been widely used as a model organism for studying cancer an integrated tool for detecting a significant change in the redox potential would ease this research, especially because a rising NADH level is seen as a hallmark of carcinogenesis.
Another prospect is preliminary research of gene therapy with DNA coding for the REX-activator protein controlling the transcription of a cancer drug. As cancer cells have extreme NAD+/NADH levels, the drug would mostly be expressed in cancer cells, leaving most normal cells unharmed.

Reference
Bonde, Mads (2009). Towards improvement of de novo biosynthesis of [confidential] by using a synthetic biology approach. Bachelor thesis, Technical University of Denmark.