Further proceeding towards malonate based dependency

The malonate-based dependency system is a promising tool in order to guarantee the fitness of the invaded S. cerevisiae cells due to the fact that malonyl-CoA is essential for its viability. If successfully transformed into S. cerevisiae, this strain has to be transformed with the acc1 knockout construct that we designed. In case the knockout was successful, we could measure the efficiency of the alternative malonyl-CoA synthesis pathway that we established by simply reducing the malonate concentration in the media step by step, to the point where the yeast cells are no longer viable without the engineered pathway.

As soon as both plasmids, the expression plasmid in S. cerevisiae and the operon plasmid in E. coli, are successfully transformed the first tests could be performed. Due to the fact, that the expression plasmid carries the mae1 gene from S. pombe, that encodes for a permease which allows the uptake of malonic acid [1], both strains can be tested in co-culture as well as in our system. This can help to compare co-culture to endosymbiosis regarding metabolism and growth rates of the symbionts. Furthermore, it will help to actually verify the advantages of endosymbiosis compared to co-culture in respect to future industrial applications.

By optimizing and avoiding bottlenecks in the pathway, the yield of the final product can be increased. For instance, a deletion of the ydfG gene in the invading strain could increase the yield of produced malonic acid [2]. This is due to the fact, that oxidation of malonic semialdehyde to malonic acid, catalyzed by yneI, directly competes with the reduction of malonic semialdehyde to 3- hydroxypropionic acid, catalyzed by the ydfG protein from E. coli.

Additionally, the malonic acid producing E. coli strain can be used in future attempts to establish a production interaction between E. coli and S. cerevisiae, since malonic acid is listed as one of the top 30 chemicals which can be derived from biomass by the US DOE [3].

Further proceeding towards protein based dependency

Unlike the expression of the YebF fusion protein, the knockout for fliC and fliD seemed to work with AB330. We will attempt to transform this knock out with the tet-inducible expression construct and test it for protein expression and export. If this is successful, further attempts towards the export of essential yeast genes will be made. For this, we also have to knock out the corresponding genes in yeast. A conditional knockout seems to be most promising, since it can be easily induced and utilized as a control.