Evaluating temperature-induced regulation of a ROSE-like RNA-thermometer for heterologous rhamnolipid production in Pseudomonas putida KT2440

Abstract

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.

Exploring the synthetic biology potential of bacteriophages for engineering non-model bacteria

“…In contrast, these parts are not yet exploited as regulatory SynBio tools for non-model hosts. Moreover, the first riboswitches and RNA thermometers have only recently been characterized in vivo in non-traditional bacteria 70 – 73 , except for B. subtilis for which riboswitch research is more mature 69 . The trans -acting counterparts of riboswitches are called translation riboregulators or antisense RNAs and can be used for multiplexed genome-wide knock-down of gene expression 39 , but their utilization as biological parts in non-traditional hosts is still limited.…”

Leveraging Pseudomonas Stress Response Mechanisms for Industrial Applications

“…P. aeruginosa is one of the most competent rhamnolipid producers and safe methods to produce rhamnolipids are being explored including strain engineering to diminish pathogenicity (Chong and Li, 2017). P. putida KT2440 rhamnolipid production increased by more than 60% when growth temperature was increased from 30 to 37 • C (Noll et al, 2019). These studies demonstrate the potential to design a process for rhamnolipid production linked to temperature and ROSE regulation.…”

Heterologous Rhamnolipid Biosynthesis: Advantages, Challenges, and the Opportunity to Produce Tailor-Made Rhamnolipids

“…Especially for applications in the (microbial) enhanced oil recovery, rhamnolipids were produced in E. coli using the common T7 expression system (Wang et al, 2007;Han et al, 2014;Jafari et al, 2014;Du et al, 2017), while others established a constitutive expression of rhlAB in E. coli (Kryachko et al, 2013). More frequently, P. putida KT2440 wild type or engineered strains were used as heterologous host for rhamnolipid biosynthesis using an inducible tac-promoter or constitutive expressed and partly synthetic promoters (Wittgens et al, 2011(Wittgens et al, , 2017Wittgens, 2013;Behrens et al, 2016;Beuker et al, 2016a,b;Tiso et al, 2016Tiso et al, , 2017Tiso et al, , 2020Anic et al, 2017Anic et al, , 2018Noll et al, 2019; Table 1). Besides well-known short-chain rhamnolipids from P. aeruginosa also the heterologous production of long chain rhamnolipids was established in this organism by expressing rhlAB and rhlC from B. glumae .…”