Autodisplay of Burkholderia gladioli carboxylesterase EstA on Pseudomonas putida KT2440

Sichwart S, Teese MG, Jose J

Abstract

The Gram-negative soil bacterium Pseudomonas putida has great potential for the biotechnological industry due to its low cultivation requirements, tolerance towards pHand temperature fluctuations, and susceptibility to genetic modifications (Poblete-Castro et al., 2012). Additionally, P. putida exhibits resistance towards various organicsolvents, which opens up the possibility of applying this organism in reactions that involve water-insoluble substrates or products and therefore require the use of organicsolvents. This is of particular relevance for ester hydrolysis reactions catalyzed by lipases and carboxylesterases (Bornscheuer, 2002). On industrial scales, the use of theseenzymes is often limited due to their costly production and purification, combined with mostly low operational stabilities under the required reaction conditions. Cell surfacedisplay, meaning the expression of an enzyme in conjunction with accessory components that mediate its transport and attachment to the surface of the host cell, couldpossibly overcome these limitations: The displayed enzyme is directly available on the host cell, thus purification steps can be omitted, and the cellular environment of thehost cell enhances enzyme stability (Jose et al., 2012). We therefore used Autodisplay, a technique that utilizes the autotransporter secretion pathway, to display thecarboxylesterase EstA from B. gladioli (Schultheiss et al., 2002) on the surface of P. putida. Surface localization of EstA could be verified by FACS, and activity assays withvarious substrates were performed to demonstrate the functionality of the enzyme. Moreover, the influence of temperature, pH, and different organic solvents on enzymeactivity was evaluated.