The microbial production of rhamnolipids has been in the focus of research for the last decades. Today, mainly heterologous production systems are targeted due to the advantage of non-pathogenic hosts as well as uncoupling from complex quorum sensing regulatory networks compared to their natural producer Pseudomonas aeruginosa. In the recent past, the presence and function of a ROSE-like RNA-thermometer located in the 5'UTR of the rhamnosyltransferase genes rhlAB has been reported in wild type P. aeruginosa. In this study, the temperature-induced regulation of this native RNA-thermometer for heterologous rhamnolipid production was evaluated and its potential application for process control is discussed. For this purpose, the non-pathogenic production host P. putida KT2440 containing the rhlAB genes with the native P. aeruginosa 5'-UTR region was used. The system was evaluated and characterized regarding the effect of temperature on growth and product formation, as represented by efficiency parameters and yields. Experimental data suggests a major effect of temperature on specific rhamnolipid production rates. With maximum values of 0.23 g/(g h) at 37 °C, this constitutes a more than 60% increase compared to the production rate of 0.14 g/(g h) at the growth optimum of 30 °C. Interestingly however, control experiments unveiled that besides the regulatory effect of the RNA-thermometer, multiple metabolic effects may contribute equally to the observed increase in production rate. As such, this work constitutes an important step towards the utilization of temperature-based process designs and enables the possibility for novel approaches for process control.
Keywords: Biosurfactant; Heterologous rhamnolipid biosynthesis; Pseudomonas putida; RNA thermometer; Thermoregulation.