Establishing the ethanologenic bacteria Zymomonas mobilis and Zymobacter palmae as platforms for surface display applications

Tozakidis IEP, Meyers A, Jose J

Abstract

Surface display technologies have emerged as valuable tools in diverse applications ranging from biosensors over vaccine delivery to high throughput library screenings. In the field of biocatalysis, surfacedisplayed enzymes offer the striking advantage of having direct access to their substrates upon expression, so that the producing organism can be employed as a whole without the need of preceding celllysis and enzyme purification steps. In contrast to secreted enzymes, surface displayed enzymes are to some extent protected by the membrane surrounding of the host organism and can more easily beseparated from the reaction due to their linkage to the microbial cells. However, a breakthrough of this technique in the chemical and biotechnological industry has not yet occurred. One reason for thisappears to be the lack of appropriate host organisms that possess valuable metabolic characteristics as well as resistance towards rough industrial handling. In this regard, the Gram negative bacteriaZymomonas mobilis and Zymobacter palmae have attracted interest due to their high ethanol productivity. A functioning surface display system for these organisms has not been reported so far. Wetherefore investigated if the autotransporter secretion pathway can be exploited for this purpose and used it to display two industrially relevant enzymes, namely carboxylesterase EstA from Burkholderiagladioli and endocellulase Cel5A from Bacillus subtilis. The expression and translocation of the respective autotransporter fusion proteins into the outer membrane was detected via SDS-PAGE. FACS,whole cell ELISA and activity assays were used to confirm both the surface exposure and functionality of the displayed enzymes. A quantitative comparison showed that Z. palmae cells exhibited higherenzyme activity, whereas Z. mobilis display seemed to be more stable.